Antibodies With High Affinity For Alpha-Klotho

ABSTRACT

An antibody and/or binding fragment thereof, wherein the antibody and/or binding fragment thereof specifically binds to an epitope of a αKlotho polypeptide, optionally a folded αKlotho or optionally with a dissociation constant (K D ) of about 2 nM or less, as measured by competitive ELISA assay, methods of making and using to diagnose kidney diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/500,478, filed on Jan. 30, 2017, which is a national phase entry ofPCT/CA2015/050728, filed on Jul. 31, 2015, which claims the benefit of35 U.S.C. § 119 based on the priority of U.S. Provisional PatentApplication No. 62/031,477, filed Jul. 31, 2014, which are incorporatedherein by reference in their entireties.

This invention was made in part with U.S. Government support under NIHGrant Nos. R01DK091392, R01DK092461 and R01DE13686. The U.S. Governmentmay have certain rights in this invention.

FIELD

An antibody and/or binding fragment thereof that specifically binds toan epitope in αKlotho polypeptide, for example with a dissociationconstant (K_(D)) of about 2 nM or less, as measured by competitive ELISAassay, as well as methods of making and using said antibody for exampleto diagnose kidney diseases.

BACKGROUND

The klotho gene was originally identified as a suppressor of prematureaging [1, reviewed in 2]. Klotho is a single-pass transmembrane proteinexpressed predominantly in kidney, the parathyroid gland, and thechoroid plexus [1, 3, 4]. Paralogous proteins with distinct functionsand expression profiles, termed βKlotho and γKlotho [5, 6] are alsoknown.

αKlotho has diverse effects including regulating ion transport, Wnt andinsulin signaling, renin-angiotensin system, recruitment of stem cells,anti-carcinogenesis, anti-fibrosis, and antioxidation. The highest levelof expression of αKlotho is in the kidney [1, 7, 8]. In addition to itstransmembrane form which is a co-receptor for fibroblast growth factor(FGF) 23,[9-11] αKlotho is also released into the circulation, urine,and cerebrospinal fluid as an endocrine substance[7, 12, 13] generatedby transcript splicing into a truncated peptide[2] or proteolyticrelease by secretases.[14, 15] A substantial portion of the circulatingαKlotho is nephrogenic in origin[16]. The phenotypic similaritiesbetween genetic αKlotho ablation and chronic kidney disease (CKD)support the notion that reduced renal expression of αKlotho ispathogenic[1, 16].

Reduced renal αKlotho transcript or protein levels[12,18-24] and serumαKlotho concentration[12, 20] was demonstrated in rodent CKD fromnephron reduction surgery, ischemia reperfusion injury, immune complexglomerulonephritis, polygenic or hormonal hypertension, metabolicsyndrome, and diabetes.[12, 18-24] This convergence suggests thatαKlotho deficiency may be a generic consequence of nephron loss. αKlothoreduction is potentially a sensitive and early biomarker of CKD and alsoprognostic of CKD complications [22]. Restoration of αKlotho inexperimental CKD in rodents ameliorates the kidney disease andextra-renal complications [12, 22, 23]. αKlotho deficiency has also beendocumented in acute kidney injury (AKI) in both rodents and humans [25].αKlotho can potentially serve as an early biomarker for AKI as it isreduced much earlier than changes in the current known biomarkers of AKI[26].

αKlotho forms a constitutive binary complex with FGF receptors (FGFRs)to confer selective affinity to FGF23 [10, 27]. Defects in αKlothoexpression result in FGF23 resistance and phosphate retention in mice[1, 28] and humans [29]. Therefore, αKlotho and FGF23 have emerged asessential components of the bone-kidney endocrine axis that regulatesphosphate metabolism [30, 31].

The extracellular domain of the membrane-anchored form of αKlotho can besecreted as a soluble protein. The soluble form is generated from themembrane-anchored form by membrane-anchored proteases and is releasedinto blood and urine [13, 15]. As noted above, membrane-anchored αKlothofunctions as part of the FGF23 receptor complex, whereas secretedαKlotho functions as an endocrine factor that exerts actions on distantorgans to exert highly pleiotropic actions as stated above (regulatingion transport, Wnt and insulin signaling, renin-angiotensin system,recruitment of stem cells, anti-carcinogenesis, anti-fibrosis, andantioxidation) [7].

Advanced CKD (Stages 4-5), characterized by kidney damage and decreasedkidney function, affects an estimated 2.6 million Canadians, greaterthan 7% of the population. A recent analysis of National VitalStatistics Report, National Health and Nutrition Examination Surveys andUS Renal Data System showed that the lifetime risks for white men, whitewomen, black men, and black women, respectively: CKD stage 3a+, 53.6%,64.9%, 51.8%, and 63.6% [84]. The impact and burden of CKD and itsassociated complications on people's lives and the health care system issignificant and will worsen in coming years [32-34]. Current approachesto treat CKD include modification of risk factors by diet andmedication, and for end stage renal disease (ESRD) by dialysis, andorgan replacement. There is an urgent need for additional therapies to,arrest or delay progression of CKD at early stages, before complicationsarise. The majority of the complications of CKD are embraced within theentity of CKD-mineral bone disturbance (CKD-MBD) which are tied todisturbances of mineral metabolism. Phosphate retention is universallyobserved in CKD patients and associated with poor outcome [35, 36].Hyperphosphatemia is usually detected only in advanced stages of CKD,when the disease is destined to progress to end-stage [37].

Recently, it has been discovered that reduced renal αKlotho expressionis one of the earliest events in CKD [12].

At present, there are some αKlotho antibodies and diagnostic kitsavailable on the market, but the existing αKlotho antibodies are not ofsufficient specificity and not efficient at immunoprecipitating αKlothofrom human serum, and the current immune-based assays for αKlotho arecostly and inadequate in sensitivity and specificity.

Low αKlotho transcript and protein levels have been described in humankidney from nephrectomy samples of end stage kidneys and biopsies frompatients with CKD [21,38]. Studies using an immune-based assay haveshown widely disparate results in terms of absolute values of serumαKlotho concentration (100-fold span in levels from different labs) anddirection of change (increased, decreased, or no change) with CKD andage[21,39-60]. The discrepant database has thwarted progress andincapacitated the ability to determine whether the promising rodent datacan be translated into meaningful human application. In addition to CKD,acute kidney injury (AKI) from a variety of causes is also associatedwith rapid decrease of αKlotho in the kidney[25, 61-65] and serum inrodents and in urine in humans[25]. There is no data on human serumαKlotho in AKI to date. There is a need for an early, sensitive, and/orspecific marker for renal injury in humans[66].

Generating antibodies to conserved proteins is challenging, as animalimmunization methods for antibody development are subject to mechanismsthat protect against auto-immunity. Synthetic antibody technology offersa powerful alternative because it is applied under defined in vitroconditions, uses antibody libraries that have not been subjected totolerance selection that remove self-reactive antibodies, and is provento yield antibodies with high affinities and specificities[67-71].Within an optimized antibody framework, sequence diversity is introducedinto the complementary determining regions (CDR's) by combinatorialmutagenesis. These libraries are coupled with phage display, with eachphage particle displaying a unique antigen-binding fragment (Fab) on itssurface while carrying the encoding DNA internally, thus achievingdirect phenotype-genotype relations. Fab-displaying phage that bind toan antigen of interest are enriched using binding selections withpurified antigens on solid support. The CDR's of binding phage clonesare identified by DNA sequencing and the Fab proteins are purified frombacteria, or converted to the full-length IgG in mammalian cells.

SUMMARY

This present disclosure relates to an antibody and/or binding fragmentthereof that binds specifically to αKlotho protein.

An aspect includes an isolated or purified antibody and/or bindingfragment thereof, wherein the antibody and/or binding fragment thereofspecifically binds to an epitope of a αKlotho polypeptide with adissociation constant (K_(D)) of about 2 nM or less, as measured bycompetitive ELISA assay.

In an embodiment, the αKlotho polypeptide specifically bound by theantibody is a folded αKlotho polypeptide.

A further aspect is antibody and/or binding fragment thereof comprisinga light chain variable region and a heavy chain variable region, thelight chain variable region comprising complementarity determiningregion CDR-L3 and the heavy chain variable region comprisingcomplementarity determining regions CDR-H1, CDR-H2 and CDR-H3, with theamino acid sequences of said CDRs comprising one or more of thesequences set forth below:

a. CDR-L3; (SEQ ID NO: 1) X₁X₂X₃X₄PX₅,

-   -   wherein X₁ is A or S, X₂ is G or A, X₃ is Y or F, X₄ is S or A,        X₅ is I or V;

b. CDR-H1: (SEQ ID NO: 2) X₆X₇X₈X₉X₁₀X₁₁,

-   -   wherein X₆ is I or V, X₇ is S or A, X₈ is Y, F or S, X₉ is Y, F        or S, X₁₀ is S or A and X₁₁ is I or V;

c. CDR-H2; (SEQ ID NO: 3) X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁,

-   -   wherein and X₁₂ is Y, F or S, X2₁₃ is I or V, X₁₄ is S or A, X₁₅        is P or S, X₁₆ is S or A, X₁₇ is Y or F, X₁₈ is G or A, X₁₉ is Y        or F, X₂₀ is T or S and X₂₁ is S, A or Y; and/or

d. CDR-H3: (SEQ ID NO: 4) X₂₂X₂₃VYX₂₄X₂₅X₂₆X₂₇WX₂₈GX₂₉GM,

-   -   wherein X₂₂ is Y or F, X₂₃ is Y or F, X₂₄ is A or S, X₂₅ is S or        A, X₂₆ is H or N, X₂₇ is G or A, X₂₈ is A or S and X₂₉ is Y or        F.

Another aspect includes a nucleic acid encoding an antibody and/orbinding fragment thereof described herein.

A further aspect is a vector comprising a nucleic acid described herein.

Another aspect includes a recombinant cell producing an antibody and/orbinding fragment thereof, nucleic acid or vector described herein.

Another aspect is an immunoassay comprising or using the antibody and/orbinding fragment thereof described herein.

Other aspects include a method for producing an antibody and/or bindingfragment thereof with specific binding affinity to an epitope of anαKlotho polypeptide described herein, an assay for measuring level ofαKlotho polypeptide in a sample, an assay for detecting and/or measuringsoluble αKlotho polypeptide as well as methods for screening, fordiagnosing or for detecting kidney condition selected from chronickidney disease (CKD) and acute kidney injury (AKI) in a subject andmethods of prognosticating disease progression and/or recovery.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the present disclosure will now be described inrelation to the drawings in which:

FIG. 1 shows the sequence, specificity and affinity of sb106 (A) CDRsequences for anti-αKlotho sb106 in the IMGT numbering scheme. (B)Specificity determination of anti-αKlotho sb106 by Fab-phage ELISA:sb106 Fab-phage were incubated with the following immobilized antigens:a complex of FGFR1c/αKlotho complex (aKL-R1), FGFR1c alone (R1), humanαKlotho (Hu aKL) and mouse αKlotho (Mu aKL), or neutravidin (NA) andbovine serum albumin (BSA) as negative controls. After washing offunbound phage, bound phages were detected using an HRP-conjugatedanti-phage antibody. Colorimetric HRP reagents allow for absorbancereadings at 450 nm. (C) Estimation of affinity by competitive Fab-phageELISA. sb106 Fab-phage were pre-incubated with 50, 5, 0.5, 0.05, 0.005and 0.0005 nM soluble human αKlotho. The binding signals to immobilizedhuman αKlotho reported are an average of two data sets. The reduction inbinding to immobilized αKlotho is indicative of the fraction bound tosoluble αKlotho, thus a 50% reduction in signal occurs when the solubleαKlotho concentration is approximately equal to the K_(D) of theinteraction.

FIG. 2 shows the characterization of sb106-Fab by immunoblot,immunohistochemistry and immunocytochemistry (A) Immunoblot of kidneylysate from wild type mice (VVT), homozygous αKlotho hypomorphic mice(kl/kl) and transgenic αKlotho overexpressing mice (Tg-KI), using themonoclonal antibody KM2076 or the sb106-Fab. GAPDH: Glyceraldehydephosphate dehydrogenase (B) Immunoblot of lysates from normal rat kidney(NRK) cells, human embryonic kidney (HEK) cells, and HEK cellstransfected with a plasmid for over-expression of αKlotho, using themonoclonal antibody KM2076 or the sb106-Fab. (C) Fresh or fixed ratparathyroid tissue probed with phalloidin for β-actin or sb106-IgG. (D)sb106 immunostaining of HEK293T cells transfected with a vector control,or vector for over-expression for αKlotho or βKlotho. Representativecells are shown. The DAPI nuclear staining is labeled “N”. (scale bar,10 μm). αKlotho staining with Fab sb106 was only observed in cellstransfected with αKlotho and not in cell transfected with βKlotho. FIG.3 shows the characterization of sb106-Fab by immunoprecipitation. (A)HEK293 cells were transfected with empty vector or varying quantities(μg/dish) of vector for expression of transmembrane full length αKlotho(TM-αKlotho) or soluble extracellular domain of αKlotho with aC-terminal FLAG epitope (s-αKlotho-FLAG). Cell lysates or cell culturemedium was immunoprecipitated (IP) with either sb106-Fab or anti-FLAGMAb. Immunocomplexes were resolved by SDS-PAGE and immunoblotted (IB)with monoclonal anti-αKlotho antibody KM2076. (B) Urine from rat, mouse,or human was immunoprecipitated with sb106-Fab, resolved by SDS-PAGE andimmunoblotted (IB) with KM2076 (left three lanes). Size-selected urine(100 kDa cut-off) was directly subjected to SDS-PAGE and immunoblotted(right three lanes). (C) Immunoprecipitations of endogenous αKlotho fromserum. Serum samples from wild type (WT) mouse, klotho^(−/−) mouse,normal human, and dialysis patient (ESRD) where incubated with sb106-Fabovernight at 4° C. Sepharose beads conjugated with anti-FLAG antibodywere then added and incubated for 2 hours at 4° C. The beads were washedand bound proteins were eluted with 2×SDS sample loading buffer.Immunoblot was performed KM2076 followed by a standard anti-rat IgGsecondary for visualization.

FIG. 4 shows the validation of IP-IB assay using human serum spiked withrecombinant αKlotho. (A) Known amounts of soluble human αKlothoectodomain were added to sera from a healthy volunteer or an anephricdialysis patient (CKD patient). αKlotho was measured in the sera usingthe IP-IB assay. (B) Similar experiment as in (A) except comparisonswere made where protease inhibitors (AEBSF 0.1 mM, aprotinin 0.3 μM,bestatin 10 μM, E-64 1 μM, leupeptin 50 μM, pepstatin A 1 μM) wereeither included or excluded from the IP. (C) αKlotho levels determinedby IP-IB (y-axis) were plotted against the added recombinant αKlotho(x-axis) in the four conditions described above. Extrapolation to zerospiking shows the level of endogenous αKlotho in the serum treated withprotease inhibitors. Only one line is shown for healthy serum with orwithout protease inhibitors as the results were indistinguishable.

FIG. 5 shows IP-IB assay of serum αKlotho in humans with chronic kidneydisease. (A) αKlotho was measured by the IP-IB assay in human sera fromnormal healthy volunteers and patients from a CKD clinic and dialysisunit using the conventional numerical staging using recombinant αKlothoas a calibration curve. Bars and error bars denote means and standarddeviations. The data was analyzed by ANOVA followed byStudent-Newman-Keuls test for pairwise multiple comparisons. P valuesachieving statistical significance between groups are indicated abovethe brackets. The number of subjects in each group is indicated at thebottom. (B) The concentrations of αKlotho in a large variety of humansera were determined either by IP-IB (x-axis) or by a commercial ELISA(y-axis) in the same samples. The dotted line represents identity. Thegrey diamonds represent sera that have been through one or morefreeze-thaw cycles (stored) and the black diamonds represent sera thawedonly once (fresh). (C) Sera from human subjects were assayed by IP-IBand ELISA. The same sera were subjected to the indicated cycles ofrepeated freeze-thaw and then assayed. Results for each sample wereexpressed as a percentage of the reading from the same sample thawedonly once. The black lines denote the mean of the different subjects.

FIG. 6 shows human urinary αKlotho levels. αKlotho was measured in theurine of healthy volunteers or patients with chronic kidney diseasestage 5 (CKD5). (A) A representative IP-IB assay using recombinantmurine αKlotho (rMKI) as a calibration with four subjects in each groupunder steady state conditions. Equal amounts of urine creatinine wereused for IP-IB. (B) Summary of the data from the IP-IB assay and thecommercial ELISA. Bars and error bars represent mean and standarddeviation from eight subjects in each group. The mean of the healthyvolunteers was set as a reference of 100%.

FIG. 7 shows SPR sensorgrams with sb106-Fab (A) SPR sensorgramillustrating binding of sb106-Fab (Fsb106) to the αKlotho-FGFR1ccomplex. The binary complex of murine αKlotho ectodomain and humanFGFR1c ligand-binding domain was immobilized on a biosensor chip and 100nM of Fsb106 were injected over the chip. Note that the Fsb106dissociates extremely slowly from the αKlotho-FGFR1c complex. (B)Overlay of SPR sensorgrams showing that sb106-Fab does not inhibitternary complex formation between FGF23, αKlotho, and FGFR1c. 10 nM ofαKlotho-FGFR1c complex alone and a mixture of 10 nM of αKlotho-FGFR1ccomplex and 100 nM of Fsb106 were injected over a biosensor chipcontaining immobilized FGF23.

FIG. 8 is a schematic of amino acid sequences of sb106. (A) Light chainsequence (SEQ ID NO: 11) of sb106. (B) Heavy chain sequence—Fab (SEQ IDNO: 12). (C) Heavy chain sequence—IgG1 (SEQ ID NO: 13). (D) Heavy chainsequence—IgG4 (SEQ ID NO: 14). Underlined amino acids are CDR sequences;bold amino acids are variable CDR sequences (L3, H1, H2 & H3); anditalicized amino acids are constant domains.

DETAILED DESCRIPTION OF THE DISCLOSURE I. Definitions

The term “αKlotho” or “alphaKlotho” as used herein refers to all knownand naturally occurring αKlotho molecules including, full length αKlothoprotein, fragments thereof such as ectodomain fragments, as well asnucleic acids encoding said protein and fragments, as determinable fromthe context used. Included are the soluble forms of αKlotho(proteolytically cleaved as well as alternatively spliced forms αKlotho,referred to as “soluble αKlotho” when present in a biological fluid suchas blood or a fraction thereof, urine or cerebrospinal fluid and havinga molecular weight of about 130 kDa, as well as the membrane-anchoredform of αKlotho, and including but not limited to mammalian αKlotho suchas human αKlotho, or rodent αKlotho including for example mouse and ratαKlotho.

The term “acute kidney injury” or “AKI” as used herein refers to anabrupt and sustained loss of kidney function for example that can leadto accumulation of urea and other chemicals in the blood, that developswithin for example seven days of an insult. AKI may be caused bydisease, injury such as crushing injury to skeletal muscle andmedication. AKI is classified in stages varying from risk (glomerularfiltration rate (GFR) decreased by 25%), injury (GFR decreased by 50%),failure (GFR decreased by 75%), loss (complete loss of kidney functionfor more than four weeks) and end-stage renal disease (complete loss ofkidney function for more than three months). AKI can be asymptomatic.

The term “early acute kidney injury” as used herein means prior to risesin serum creatinine.

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified amino acids.

The term “antibody” as used herein is intended to include humanantibodies, monoclonal antibodies, polyclonal antibodies, single chainand other chimeric antibodies. The antibody may be from recombinantsources and/or produced in transgenic animals. The antibody in anembodiment comprises a heavy chain variable region or a heavy chaincomprising a heavy chain complementarity determining region 1, heavychain complementarity determining region 2 and heavy chaincomplementarity determining region 3, as well as a light chain variableregion or light chain comprising a light chain complementaritydetermining region 1, light chain complementarity determining region 2and light chain complementarity determining region 3.

The term “binding fragment” as used herein is intended to includewithout limitations Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers,minibodies, diabodies, and multimers thereof, multispecific antibodyfragments and Domain Antibodies. Antibodies can be fragmented usingconventional techniques. For example, F(ab′)2 fragments can be generatedby treating the antibody with pepsin. The resulting F(ab′)2 fragment canbe treated to reduce disulfide bridges to produce Fab′ fragments. Papaindigestion can lead to the formation of Fab fragments. Fab, Fab′ andF(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecificantibody fragments and other fragments can also be synthesized byrecombinant techniques.

A “conservative amino acid substitution” as used herein, is one in whichone amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties. Suitableconservative amino acid substitutions can be made by substituting aminoacids with similar hydrophobicity, polarity, and R-chain length for oneanother. Examples of conservative amino acid substitution include:

Conservative Substitutions Type of Amino Acid Substitutable Amino AcidsHydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr Sulphydryl CysAliphatic Val, Ile, Leu, Met Basic Lys, Arg, His Aromatic Phe, Tyr, Trp

The term “control” as used herein refers to a sample from a subject or agroup of subjects who are either known as having a kidney disease or nothaving the disease, and/or a value determined from said group ofsubjects, wherein subjects with an αKlotho level at or below such valueare likely to have the disease. The disease can be for example chronickidney disease (CKD) or acute kidney injury (AKI). The disease can alsobe for example a stage of CKD such as stage 1 CKD, stage 2 CKD, stage 3CKD, stage 4 CKD or stage 5 CKD; higher stage being more severe. Inaddition, the control can be for example derived from tissue of the sametype as the sample of the subject being tested. In methods directed tomonitoring, the control can also be tissue from the same subject takenat different time point for example the control can be a sample from thesame subject taken prior to a treatment for a kidney disease.

The term “chronic kidney disease” or “CKD” refers to a disease causing aprogressive loss in renal function. CDK is classified according to fivestages which are determined according to a defined glomerular filtrationrate (GFR). Stage 1 CKD is defined by a GFR of ≥90 mL/min/1.73 m², stage2 CDK is defined by a GFR between 60-89 mL/min/1.73 m², stage 3 CKD isdefined by a GFR between 30-59 mL/min/1.73 m², stage 4 CKD is defined bya GFR between 15-29 mL/min/1.73 m² and stage 5 CKD is defined by a GFRof less than 15 mL/min/1.73 m². Normal kidney function is defined by aGFR between 100-130 mL/min/1.73 m² or 90 mL/min/1.73 m² withoutproteinuria.

The term “early chronic kidney disease” refers to earlier stages of CKD,and means in an embodiment stage 1 and/or stage 2 CKD are early CKD.Frequently, there are no elevations of FGF23, PTH, and phosphate.Subjects with stage 1 CKD almost never present any symptoms indicatingkidney damage. Subjects with stage 2 CKD do not necessarily presentsymptoms indicating kidney damage but occasionally do.

The term “denatured” as used herein means a polypeptide that has losttertiary and/or secondary structure (e.g. fully unfolded protein), forexample when exposed to denaturing conditions in SDS sample loadingbuffer.

The term “detectable tag” as used herein refers to moieties such aspeptide sequences that can be appended or introduced into recombinantprotein.

The term “epitope” as used herein refers to the site on the antigen thatis recognized by the antibodies or binding fragments disclosed herein.

The term “heavy chain complementarity determining region” as used hereinrefers to regions of hypervariability within the heavy chain variableregion of an antibody molecule. The heavy chain variable region hasthree complementarity determining regions termed heavy chaincomplementarity determining region 1 (CDR-H1), heavy chaincomplementarity determining region 2 (CDR-H2) and heavy chaincomplementarity determining region 3 (CDR-H3) from the amino terminus tocarboxy terminus. The numbering used herein is the IMGT numbering.

The term “heavy chain variable region” as used herein refers to thevariable domain of the heavy chain comprising the heavy chaincomplementarity determining region 1, heavy chain complementaritydetermining region 2 and heavy chain complementarity determining region3. One or more amino acids or nucleotides can be modified for examplereplaced with a conservative substitution, for example outside the CDRsequences.

The term “host cell” refers to a cell into which a recombinant DNAexpression vector can be introduced to produce a recombinant cell. Thehost cell can be a bacterial cell such as E. coli but can also be anytype of microbial, yeast, fungi, insect or mammalian host cell.

The terms “IMGT numbering” or “ImMunoGeneTics database numbering”, whichare recognized in the art, refer to a system of numbering amino acidresidues which are more variable (i.e. hypervariable) than other aminoacid residues in the heavy and light chain variable regions of anantibody, or antigen binding portion thereof (85). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 32 to 38 for CDR-H1, amino acid positions 55 to 64 for CDR-H2,and amino acid positions 107 to 117 for CDR-H3.

For light chain variable region, the hypervariable region ranges fromamino acid positions 24 to 39 for CDR-L1, amino acid positions 56 to 69for CDR-L2, and amino acid positions 105 to 117 for CDR-L3.

The term “isolated antibody or binding fragment thereof” or “isolatedand purified antibody or binding fragment thereof” refers to an antibodyor binding fragment thereof that is substantially free of cellularmaterial or culture medium when produced by recombinant DNA techniques,or chemical precursors, or other chemicals when chemically synthesizedand/or other antibodies, for example directed to a different epitope.

The term “K_(D)” refers to the dissociation constant of a complex forexample of a particular antibody-antigen interaction

The term “light chain complementarity determining region” as used hereinrefers to regions of hypervariability within the light chain variableregion of an antibody molecule. Light chain variable regions have threecomplementarity determining regions termed light chain complementaritydetermining region 1, light chain complementarity determining region 2and light chain complementarity determining region 3 from the aminoterminus to the carboxy terminus.

The term “light chain variable region” as used herein refers to thevariable domain of the light chain comprising the light chaincomplementarity determining region 1, light chain complementaritydetermining region 2 and light chain complementarity determining region3.

The term “native” or “natively folded” as used herein refers to aprotein in its native conformation (e.g. 3D conformation) or in aconformation sufficient to confer functionality, including for examplepartially unfolded protein capable of binding a receptor or ligand. Forexample, folded αKlotho protein is capable of binding to a FGF receptorsuch as FGFR1c and can form a FGFR1c: αKlotho complex.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentapplication may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine. The nucleic acid can be either double stranded or singlestranded, and represents the sense or antisense strand. Further, theterm “nucleic acid” includes the complementary nucleic acid sequences aswell as codon optimized or synonymous codon equivalents. The term“isolated nucleic acid sequences” as used herein refers to a nucleicacid substantially free of cellular material or culture medium whenproduced by recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An isolated nucleic acid is alsosubstantially free of sequences which naturally flank the nucleic acid(i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) fromwhich the nucleic acid is derived.

The term “polypeptide” as used herein refers to a polymer consisting alarge number of amino acid residues bonded together in a chain. Thepolypeptide can form a part or the whole of a protein. The polypeptidemay be arranged in a long, continuous and unbranched peptide chain. Thepolypeptide may also be arranged in a biologically functional way. Thepolypeptide may be folded into a specific three dimensional structurethat confers it a defined activity. The term “polypeptide” as usedherein is used interchangeably with the term “protein”.

The term “isolated polypeptide” as used herein means substantially freeof cellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors, or other chemicals when chemicallysynthesized.

The term “reference agent” as used herein refers to an agent that can beused in an assay and that can be for example a standard amount ofαKlotho protein used as a reference for example for detecting, screeningor for diagnosing kidney condition such as chronic kidney disease andacute kidney disease.

The term “sample” as used herein refers to any biological fluid, cell ortissue sample from a subject, which can be assayed for αKlotho such assoluble biomarkers. For example the sample can comprise urine, serum,plasma or cerebrospinal fluid. The sample can for example be a“post-treatment” sample wherein the sample is obtained after one or moretreatments, or a “base-line sample” which is for example used as a baseline for assessing disease progression.

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modifiedas in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the NBLAST nucleotide programparameters set, e.g., for score=100, wordlength=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentapplication. BLAST protein searches can be performed with the XBLASTprogram parameters set, e.g., to score-50, wordlength=3 to obtain aminoacid sequences homologous to a protein molecule of the presentinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., 1997, NucleicAcids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to performan iterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., of XBLAST andNBLAST) can be used (see, e.g., the NCBI website). Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The percent identity between twosequences can be determined using techniques similar to those describedabove, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(% (G+C)−600/l), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm—5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

The term “subject” as used herein refers to any member of the animalkingdom, preferably a mammal, more preferably a human being or a rodentsuch as a rat or a mouse. In one embodiment, the subject is suspected ofhaving a kidney disorder such as chronic kidney disease (CKD) or acutekidney injury (AKI).

The term “variant” as used herein includes one or more amino acid and/ornucleotide modifications in a sequence (polypeptide or nucleic acidrespectively) for example, one or more modifications of a light chain ora heavy chain complementarity determining region (CDR) disclosed hereinthat perform substantially the same function as the light chain andheavy chain CDRs disclosed herein in substantially the same way. Forinstance, variants of the CDRs disclosed herein have the same functionof being able to specifically bind to an epitope on the folded αKlothoprotein. In one embodiment, variants of CDRs disclosed herein include,without limitation, conservative amino acid substitutions. Variants ofthe CDRs also include additions and deletions to the CDR sequencesdisclosed herein. In addition, variant nucleotide sequences andpolypeptide sequences include analogs and derivatives thereof.

The term “level” as used herein refers to an amount (e.g. relativeamount or concentration) of αKlotho protein that is detectable ormeasurable in a sample. For example, the soluble αKlotho level can be aconcentration such as pM or a relative amount such as 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6,3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 and/or 10 times a control level, wherefor example, the control level is the level of soluble αKlotho in ahealthy subject.

In understanding the scope of the present disclosure, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. Finally, terms of degree such as “substantially”, “about”and “approximately” as used herein mean a reasonable amount of deviationof the modified term such that the end result is not significantlychanged. These terms of degree should be construed as including adeviation of at least ±5% of the modified term if this deviation wouldnot negate the meaning of the word it modifies.

In understanding the scope of the present disclosure, the term“consisting” and its derivatives, as used herein, are intended to beclose ended terms that specify the presence of stated features,elements, components, groups, integers, and/or steps, and also excludethe presence of other unstated features, elements, components, groups,integers and/or steps.

The recitation of numerical ranges by endpoints herein includes allnumbers and fractions subsumed within that range (e.g. 1 to 5 includes1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood thatall numbers and fractions thereof are presumed to be modified by theterm “about.” Further, it is to be understood that “a,” “an,” and “the”include plural referents unless the content clearly dictates otherwise.The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%,preferably 10-20%, more preferably 10% or 15%, of the number to whichreference is being made.

Further, the definitions and embodiments described in particularsections are intended to be applicable to other embodiments hereindescribed for which they are suitable as would be understood by a personskilled in the art. For example, in the following passages, differentaspects of the invention are defined in more detail. Each aspect sodefined may be combined with any other aspect or aspects unless clearlyindicated to the contrary. In particular, any feature indicated as beingpreferred or advantageous may be combined with any other feature orfeatures indicated as being preferred or advantageous.

II. Antibody and/or Binding Fragment Thereof

The present disclosure relates to an antibody and/or binding fragmentthereof and methods of making and use for example for diagnosing and/orprognosticating kidney diseases.

Accordingly, a first aspect is a an antibody and/or binding fragmentthereof, wherein the antibody and/or binding fragment thereofspecifically binds to an epitope of a αKlotho polypeptide with adissociation constant (K_(D)) of about 10 nM or less, as measured bycompetitive ELISA assay. As shown in the Examples below, competitiveELISA assays showed a dose-response curve for anti-αKlotho sb106 bindingto αKlotho, and the affinity of the interaction was estimated to bearound 1-2 nM (FIG. 1C).

In an embodiment, the αKlotho polypeptide is folded, optionally innative conformation (e.g. fully folded). As demonstrated herein, theanti-αKlotho sb106 antibody has specific binding affinity to foldedαKlotho such as natively folded αKlotho. For example, as shown inExample 5, the sb106 antibody has high binding affinity to αKlotho undernative conditions but has much weaker or no binding affinity to αKlothounder denaturing conditions.

Accordingly another aspect is an antibody and/or binding fragmentthereof, wherein the antibody and/or binding fragment thereofspecifically binds to an epitope of a folded αKlotho polypeptide.

Further, anti-αKlotho sb106 has a high binding affinity in freshlyprepared or mildly fixed cells.

Accordingly a further aspect is an antibody and/or binding fragmentthereof, wherein the antibody and/or binding fragment thereofspecifically binds to αKlotho polypeptide in an unfixed or mildly fixedsample.

In an embodiment, the αKlotho polypeptide in the unfixed or mildly fixedsample is folded αKlotho.

The CDR regions of sb106 were determined and are shown In FIG. 1A.Further homologous mutations were introduced at each amino acid positionof the SB106 CDRs, (e.g. for each position either the original aminoacid was retained or a conservative amino acid change was introduced anda new Fab-phage library was constructed. Selections were performed usingthe new library using the alphaKlotho-FGFR1c complex as an antigen.Clones that bound to the antigen were isolated and sequenced and areshown in Table 2.

Accordingly another aspect includes an antibody and/or binding fragmentthereof comprising a light chain variable region and a heavy chainvariable region, the light chain variable region comprisingcomplementarity determining region CDR-L3 and the heavy chain variableregion comprising complementarity determining regions CDR-H1, CDR-H2 andCDR-H3, one or more of said CDRs comprising an amino acid sequence asset forth below:

a. CDR-L3; (SEQ ID NO: 1) X₁X₂X₃X₄PX₅,

-   -   wherein X₁ is A or S, X₂ is G or A, X₃ is Y or F, X₄ is S or A,        X₅ is I or V;

b. CDR-H1: (SEQ ID NO: 2) X₆X₇X₈X₉X₁₀X₁₁,

-   -   wherein X₆ is I or V, X₇ is S or A, X₈ is Y, F or S, X₉ is Y, F        or S, X₁₀ is S or A and is I or V;

c. CDR-H2; (SEQ ID NO: 3) X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁,

-   -   wherein and X₁₂ is Y, F or S, X2₁₃ is I or V, X₁₄ is S or A, X₁₅        is P or S, X₁₆ is S or A, X₁₇ is Y or F, X₁₈ is G or A, X₁₉ is Y        or F, X₂₀ is T or S and X₂₁ is S, A or Y;

d. CDR-H3: (SEQ ID NO: 4) X₂₂X₂₃VYX₂₄X₂₅X₂₆X₂₇WX₂₈GX₂₉GM,

-   -   wherein X₂₂ is Y or F, X₂₃ is Y or F, X₂₄ is A or S, X₂₅ is S or        A, X₂₆ is H or N, X₂₇ is G or A, X₂₈ is A or S and X₂₉ is Y or        F.

Antibody fragments were isolated having CDR sequences as described inTable 2. In an embodiment, the antibody or binding fragment thereof hasa CDR-L3, CDR-H1, CDR-H2 and CDR-H3 selected from the SEQ ID NOs: 15-120as listed in Table 2.

In an embodiment, the antibody comprises a CDR-L3 with a sequenceselected from SEQ ID NO: 1, a CDRH1 with a sequence selected from SEQ IDNO: 2, a CDR-H2 with a sequence selected from SEQ ID NO:3 and/or aCDR-H3 selected from SEQ ID NO:4 and exhibits a K_(D) for αKlothospecific binding of about or less than 10 nM, about or less than 9 nM,about or less than 8 nM, about or less than 7 nM, about or less than 6nM, about or less than 5 nM, about or less than 4 nM, about or less than3 nM, about or less than 2 nM or about or less than 1 nM.

As mentioned, the sequences of Fab anti-αKlotho sb106 CDRL3 and CDRH1,2, 3 are shown here (FIG. 1A) and in SEQ ID NOs: 5-8. To furthercharacterize the binding specificity of a clone, the anti-αKlotho sb106antibody was assayed against a panel of individually purified components(FIG. 1B). Anti-αKlotho sb106 binds to αKlotho alone or within thecontext of the FGFR1c/αKlotho complex, and is cross-reactive to bothhuman and mouse species. Anti-αKlotho sb106 is also cross-reactive torat species.

Accordingly in another embodiment, the complementarity determiningregions comprise the amino acid sequences set forth below:

Light Chain Variable Region:

(SEQ ID NO: 5) CDR-L3: AGYSPI

Heavy Chain Variable Region:

(SEQ ID NO: 6) CDR-H1: ISYYSI (SEQ ID NO: 7) CDR-H2: YISPSYGYTS (SEQ IDNO: 8) CDR-H3: YYVYASHGWAGYGM

In a further embodiment, the light chain variable region furthercomprises complementarity determining regions CDR-L1 and/or CDR-L2comprising the amino acid sequences set forth below:

(SEQ ID NO: 9) CDR-L1: QSVSSA (SEQ ID NO: 10) CDR-L2: SAS

In another embodiment, the complementarity determining regions compriseone or more of the amino acid sequences as set forth in SEQ ID NOs: 5-8,or 15-120. In an embodiment, the CDR regions comprise a CDR-L3, CDR-H1,CDR-H2 and CDR-H3

In another embodiment, the antibody and/or binding fragment thereofcomprises a light chain with the amino acid sequence set forth below:

Light Chain Sequence:

(SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQAGYSPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

In another embodiment, the antibody and/or binding fragment thereofcomprises a heavy chain variable region with the amino acid sequence setforth below:

Heavy Chain Variable Region Sequence:

(SEQ ID NO: 12) EVQLVESGGGLVQPGGSLRLSCAASGFNISYYSIHWVRQAPGKGLEWVAYISPSYGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYYVYASHGWAGYGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

The antibody optionally a human antibody can be any class ofimmunoglobulins including: IgM, IgG, IgD, IgA or IgE; and any isotype,including: IgG1, IgG2, IgG3 and IgG4.

Humanized or chimeric antibody may include sequences from one or morethan one isotype or class.

Further, antibodies described herein may be produced as antigen bindingfragments such as Fab, Fab′ F(ab′)₂, Fd, Fv and single domain antibodyfragments, or as single chain antibodies in which the heavy and lightchains are linked by a spacer. Also, the human or chimeric antibodiesmay exist in monomeric or polymeric form.

Chimeric antibodies can be prepared using recombinant techniques. Asdescribed in the Examples, the Fab identified in the screen wasreformatted into full length IgG by subcloning the variable domains ofthe antibody's light and heavy chains into mammalian expression vectorsand producing the IgG protein for example as shown in the Examples usinghuman embryonic kidney cells (HEK293T). As described elsewhere any celltype suitable for expressing an antibody can be used.

In yet another embodiment, the antibody and/or binding fragment thereofcomprises a heavy chain IgG1 or IgG4 isotype, optionally with the aminoacid sequence of an isotype set forth below:

IgG1: (SEQ ID NO: 13) EVQLVESGGGLVQPGGSLRLSCAASGFNISYYSIHWVRQAPGKGLEWVAYISPSYGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYYVYASHGWAGYGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK IgG4:(SEQ ID NO: 14) EVQLVESGGGLVQPGGSLRLSCAASGFNISYYSIHWVRQAPGKGLEWVAYISPSYGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYYVYASHGWAGYGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSPGK

In yet another embodiment, the light chain complementarity determiningregion CDR-L3 and heavy chain complementarity determining regionsCDR-H1, CDR-H2 and CDR-H3 have at least 70%, at least 80% or at least90% sequence identity to SEQ ID NOS: 5 to 8, respectively. Specific CDRsequences are provided in SEQ ID NOs: 15-120 as shown in Table 2 whichinclude at least 70% sequence identity to SEQ ID NOs:5-8.

In an embodiment, the antibody, binding fragment thereof, optionally theCDR sequence has one or more conservative substitutions.

In yet another embodiment, the antibody comprises the light chaincomplementarity determining region CDR-L3 and heavy chaincomplementarity determining regions CDR-H1, CDR-H2 and CDR-H3 having asequence set for in SEQ ID NOS: 1 to 4, respectively, optionally withthe light chain variable region, the heavy chain variable region havingat least 70%, at least 80% or at least 90% sequence identity to SEQ IDNOs: 11 and 12 respectively or optionally in the context of a heavychain IgG1 or IgG4, having at least 70%, at least 80% or at least 90%sequence identity to SEQ ID NOs:13 and 14. For example one of more CDRsdescribed herein can be grafted into an optimized or selected antibody,antibody chain or variable region.

In one embodiment, the antibody and/or binding fragment thereof isselected from the group consisting of a an immunoglobulin molecule, aFab, a Fab′, a F(ab)2, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, adisulfide linked scFv, a single chain domain antibody, a diabody, adimer, a minibody, a bispecific antibody fragment, a chimeric antibody,a human antibody, a humanized antibody and a polyclonal antibody.

Fab, Fab′ and F(ab′)₂, scFv, dsFv, ds-scFv, dimers, minibodies,diabodies, bispecific antibody fragments and other fragments can besynthesized by recombinant techniques.

Antibodies can also be fragmented using conventional techniques. Forexample, F(ab′)₂ fragments can be generated by treating the antibodywith pepsin. The resulting F(ab′)₂ fragment can be treated to reducedisulfide bridges to produce Fab′ fragments. Papain digestion can leadto the formation of Fab fragments.

In an embodiment, the antibody is a human antibody.

Human antibodies are optionally obtained from transgenic animals (U.S.Pat. Nos. 6,150,584; 6,114,598; and 5,770,429). In this approach theheavy chain joining region (J_(H)) gene in a chimeric or germ-linemutant mouse is deleted. Human germ-line immunoglobulin gene array issubsequently transferred to such mutant mice. The resulting transgenicmouse is then capable of generating a full repertoire of humanantibodies upon antigen challenge.

In an embodiment, the antibody is a chimeric antibody comprising one ormore CDRs selected from SEQ ID NOs: 1-10 or SEQ ID NOs: 15 to 120.

As mentioned above, FIG. 10 demonstrates for example that anti-αKlothosb106 binds to αKlotho either alone or in the context of a FGFR/αKlothocomplex such as a FGFR1c/αKlotho complex. The affinity of theinteraction between anti-αKlotho sb106 and αKlotho, as measured bycompetitive ELISA assays, is in the single-digit nanomolar range(IC₅₀=1.7 nM).

In one embodiment, the antibody and/or binding fragment thereof has aK_(D) for αKlotho specific binding of about or less than 10 nM, about orless than 9 nM, about or less than 8 nM, about or less than 7 nM, aboutor less than 6 nM, about or less than 5 nM, about or less than 4 nM,about or less than 3 nM, about or less than 2 nM or about or less than 1nM.

The antibody and/or binding fragment thereof herein disclosed iscross-reactive to several species. In an embodiment, the αKlothopolypeptide bound is mammalian αKlotho polypeptide, for example, theαKlotho polypeptide is selected from human αKlotho polypeptide or rodentαKlotho polypeptide such as mouse αKlotho polypeptide or rat αKlothopolypeptide.

In another embodiment, the folded αKlotho polypeptide is soluble foldedαKlotho polypeptide. For example, the antibody and/or binding fragmentthereof binds soluble folded αKlotho polypeptide found in urine, plasma,and/or serum.

In yet another embodiment, the antibody and/or binding fragment thereofbinds a complex comprising folded αKlotho polypeptide. For example, thefolded αKlotho polypeptide forms a complex with a fibroblast growthfactor (FGF) receptor, optionally FGFR1c.

In a further embodiment, the antibody and/or binding fragment islabelled and/or conjugated to a tag, for example to produce a diagnosticagent. For example, the detectable tag can be a purification tag such asa His-tag, a HA-tag, a GST-tag, biotin or a FLAG-tag.

The label is preferably capable of producing, either directly orindirectly, a detectable signal. For example, the label may beradio-opaque or a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I,¹³¹I; a fluorescent (fluorophore) or chemiluminescent (chromophore)compound, such as fluorescein isothiocyanate, rhodamine or luciferin; anenzyme, such as alkaline phosphatase, beta-galactosidase or horseradishperoxidase; an imaging agent; or a metal ion.

Another aspect of the disclosure relates to an antibody complexcomprising the antibody and/or binding fragment thereof and αKlotho,optionally further comprising FGFR1c.

In an embodiment, the antibody complex comprises FGFR1c and optionallyfurther comprises FGF23.

In an embodiment, the antibody and/or binding fragment thereof is anisolated antibody and/or binding fragment thereof.

Yet another aspect is a nucleic acid encoding an antibody and/or partthereof such as a binding fragment thereof described herein. In anembodiment, the nucleic acid encodes an antibody and/or binding fragmentthereof comprising a light chain variable region and a heavy chainvariable region, the light chain variable region comprisingcomplementarity determining regions CDR-L1, CDR-L2 and CDR-L3 and theheavy chain variable region comprising complementarity determiningregions CDR-H1, CDR-H2 and CDR-H3, with the amino acid sequences of saidCDRs comprising one or more of the sequences set forth below:

(SEQ ID NO: 1) CDR-L3; X₁X₂X₃X₄PX₅,

-   -   wherein X₁ is A or S, X₂ is G or A, X₃ is Y or F, X₄ is S or A,        X₅ is I or V;

(SEQ ID NO: 2) CDR-H1: X₆X₇X₈X₉X₁₀X₁₁,

-   -   wherein X₆ is I or V, X₇ is S or A, X₈ is Y, F or S, X₉ is Y, F        or S, X₁₀ is S or A and is I or V;

(SEQ ID NO: 3) CDR-H2; X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁,

-   -   wherein and X₁₂ is Y, F or S, X2₁₃ is I or V, X₁₄ is S or A, X₁₅        is P or S, X₁₆ is S or A, X₁₇ is Y or F, X₁₈ is G or A, X₁₉ is Y        or F, X₂₀ is T or S and X₂₁ is S, A or Y;

(SEQ ID NO: 4) CDR-H3: X₂₂X₂₃VYX₂₄X₂₅X₂₆X₂₇WX₂₈GX₂₉GM,

-   -   wherein X₂₂ is Y or F, X₂₃ is Y or F, X₂₄ is A or S, X₂₅ is S or        A, X₂₆ is H or N, X₂₇ is G or A, X₂₈ is A or S and X₂₉ is Y or        F.

Nucleic acids encoding a heavy chain or a light chain are also provided,for example encoding a heavy chain comprising CDR-H1, CDR-H2 and/orCDR-H3 regions described herein or encoding a light chain comprisingCDR-L1, CDR-L2 and/or CDR-L3 regions described herein.

The present disclosure also provides variants of the nucleic acidsequences that encode for the antibody and/or binding fragment thereofdisclosed herein. For example, the variants include nucleotide sequencesthat hybridize to the nucleic acid sequences encoding the antibodyand/or binding fragment thereof disclosed herein under at leastmoderately stringent hybridization conditions or codon degenerate oroptimized sequences In another embodiment, the variant nucleic acidsequences have at least 70%, most preferably at least 80%, even morepreferably at least 90% and even most preferably at least 95% sequenceidentity to nucleic acid sequences encoding SEQ ID NOs: 1 to 120.

The antibodies described herein can comprise one or more of the featuresdescribed herein.

In an embodiment, the nucleic acid is an isolated nucleic acid.

Another aspect is a vector comprising the nucleic acid herein disclosed.In an embodiment, the vector is an isolated vector.

The vector can be any vector suitable for producing an antibody and/orbinding fragment thereof, including for example vectors describedherein. Possible expression vectors include but are not limited tocosmids, plasmids, or modified viruses (e.g. replication defectiveretroviruses, adenoviruses and adeno-associated viruses).

A further aspect is a recombinant cell producing the antibody and/orbinding fragment thereof herein disclosed or the vector hereindisclosed.

The recombinant cell can generated using any cell suitable for producinga polypeptide, for example suitable for producing an antibody and/orbinding fragment thereof.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins of the invention may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells.

More particularly, bacterial host cells suitable for producingrecombinant antibody producing cells include E. coli, B. subtilis,Salmonella typhimurium, and various species within the genusPseudomonas, Streptomyces, and Staphylococcus, as well as many otherbacterial species well known to one of ordinary skill in the art.Suitable bacterial expression vectors preferably comprise a promoterwhich functions in the host cell, one or more selectable phenotypicmarkers, and a bacterial origin of replication. Representative promotersinclude the β-lactamase (penicillinase) and lactose promoter system, thetrp promoter and the tac promoter.

Representative selectable markers include various antibiotic resistancemarkers such as the kanamycin or ampicillin resistance genes. Suitableexpression vectors include but are not limited to bacteriophages such aslambda derivatives or plasmids such as pBR322, the pUC plasmids pUC18,pUC19, pUC118, pUC119, and pNH8A, pNH16a, pNH18a, and Bluescript M13(Stratagene, La Jolla, Calif.).

Suitable yeast and fungi host cells include, but are not limited toSaccharomyces cerevisiae, Schizosaccharomyces pombe, the genera Pichiaor Kluyveromyces and various species of the genus Aspergillus. Examplesof vectors for expression in yeast S. cerivisiae include pYepSec1, pMFa,pJRY88, and pYES2 (Invitrogen Corporation, San Diego, Calif.). Protocolsfor the transformation of yeast and fungi are well known to those ofordinary skill in the art.

Suitable mammalian cells include, among others: COS (e.g., ATCC No. CRL1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa(e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), NS-1 cells and anyderivatives of these lines.

In an embodiment, the mammalian cells used to produce a recombinantantibody are selected from CHO, HEK293 cells or Freestyle™ 293-F cells(Life technologies). FreeStyle 293-F cell line is derived from the 293cell line and can be used with the FreeStyle™ MAX 293 Expression System,FreeStyle™ 293 Expression System or other expression systems.

Suitable expression vectors for directing expression in mammalian cellsgenerally include a promoter (e.g., derived from viral material such aspolyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), as well asother transcriptional and translational control sequences.

In an embodiment, the vector is designed for production of light chainor IgG1 heavy chain.

Suitable insect cells include cells and cell lines from Bombyx orSpodotera species. Baculovirus vectors available for expression ofproteins in cultured insect cells (SF 9 cells) include the pAc seriesand the pVL series.

The recombinant expression vectors may also contain genes which encode afusion moiety (i.e. a “fusion protein”) which provides increasedexpression or stability of the recombinant peptide; increased solubilityof the recombinant peptide; and aid in the purification of the targetrecombinant peptide by acting as a ligand in affinity purification,including for example tags and labels described herein. Further, aproteolytic cleavage site may be added to the target recombinant proteinto allow separation of the recombinant protein from the fusion moietysubsequent to purification of the fusion protein. Typical fusionexpression vectors include pGEX (Amrad Corp., Melbourne, Australia),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the recombinant protein.

“Operatively linked” is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid. Suitable regulatory sequences may be derived from avariety of sources, including bacterial, fungal, viral, mammalian, orinsect genes. Selection of appropriate regulatory sequences is dependenton the host cell chosen and may be readily accomplished by one ofordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector.

In an embodiment, expression of the antibody or binding fragment thereofis under the control of an inducible promoter. Examples of induciblenon-fusion expression vectors include pTrc (28) and pET 11d.

The recombinant expression vectors may also contain a marker gene whichfacilitates the selection of host cells transformed or transfected witha recombinant molecule of the invention. Examples of selectable markergenes are genes encoding a protein such as G418 and hygromycin whichconfer resistance to certain drugs, β-galactosidase, chloramphenicolacetyltransferase, firefly luciferase, or an immunoglobulin or portionthereof such as the Fc portion of an immunoglobulin preferably IgG.Transcription of the selectable marker gene is monitored by changes inthe concentration of the selectable marker protein such asβ-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. If the selectable marker gene encodes a protein conferringantibiotic resistance such as neomycin resistance transformant cells canbe selected with G418. Cells that have incorporated the selectablemarker gene will survive, while the other cells die. This makes itpossible to visualize and assay for expression of recombinant expressionvectors of the invention and in particular to determine the effect of amutation on expression and phenotype. It will be appreciated thatselectable markers can be introduced on a separate vector from thenucleic acid of interest. Other selectable markers include fluorescentproteins such as GFP which may be cotransduced with the nucleic acid ofinterest.

Yet another aspect is a composition comprising the antibody and/orbinding fragment thereof, the nucleic acid herein disclosed or therecombinant cell herein disclosed, optionally in combination with asuitable diluent or carrier.

The composition can be a lyophilized powder or aqueous or non-aqueoussolution or suspensions, which may further contain antioxidants,buffers, bacteriostats and solutes. Other components that may be presentin such compositions include water, surfactants (such as Tween),alcohols, polyols, glycerin and vegetable oils, for example.

Suitable diluents for nucleic acids include but are not limited towater, saline solutions and ethanol.

Suitable diluents for polypeptides, including antibodies or fragmentsthereof and/or cells include but are not limited to saline solutions, pHbuffered solutions and glycerol solutions or other solutions suitablefor freezing polypeptides and/or cells.

The composition can further comprise stabilizing agents, for examplereducing agents, hydrophobic additives, and protease inhibitors whichare added to physiological buffers.

In an embodiment, the polypeptide is comprised in the solution at aconcentration of about 0.5 mg/mL or higher.

Another aspect is an immunoassay comprising the antibody and/or bindingfragment thereof herein disclosed as further described below.

In an embodiment, the immunoassay is an enzyme linked immunosorbentassay (ELISA). For example, the ELISA is a sandwich ELISA. In anembodiment the assay is a proximity ligation assay (PLA). In anembodiment, the assay is an immunoprecipation optionally combined withimmunoblot detection. In an embodiment, the immunoassay is combined witha mass spectrophometric assay. In an embodiment, the immunoassay is aparticle-based flow cytometric assay.

As described in the Examples, the anti-αKlotho sb106 antibody recognizesboth the cellular bound and unbound forms of αKlotho. In cell bindingstudies, anti-αKlotho sb106 bound to HEK293T cells expressing αKlothobut not its paralog βKlotho (FIG. 2A). The anti-αKlotho sb106 antibodycan also immunoprecipitate unbound αKlotho from both human and mouseserum (FIG. 2B). This antibody can immunopreciptate αKlotho with highaffinity and specificity devoid of additional bands.

In an embodiment, the immunoassay is a high throughput diagnostic assay.

III. Methods

Another aspect of the disclosure is a method for producing antibodyand/or binding fragment thereof with specific binding affinity to anepitope of a folded αKlotho polypeptide.

In one embodiment, the antibody is isolated from an antibody library.For example, the antibody library can be an antibody phage-displaylibrary. In one embodiment, the antibody phage-display library is ahuman Fab phage-display library.

As described below, high throughput, phage-display technology was usedto generate the anti-αKlotho sb106 antibody described in the Examplesbelow. Phage-displayed synthetic antibody libraries were screened withthe antigen using established methods [67,75]. Antibody binders(phage-displayed or purified) were tested by ELISAs. Purified antigensfor the primary screen and subsequent ELISAs included mouse αKlotho,human FGFR1c or a human FGFR1c/mouse αKlotho complex and were producedor purchased from R&D Systems (human αKlotho). As described in theExamples, the sequence of the Fab's antibody-binding region(complementarity determining regions, or CDRs, of the antibody light andheavy chains) was decoded from the DNA carried by the unique bindingphage. The CDR regions randomized in the synthetic antibody library usedwere light chain 3 (CDR-L3) and heavy chain 1, 2, and 3(CDR-H1,-H2,-H3). An anti-αKlotho antibody that binds αKlotho with forexample at least a binding affinity of 10 nM or 2 nM or less can beisolated using a phage library as described.

In an embodiment, the method further comprises randomizing CDR-L1 and/orCDR-L2.

As described in the Examples below, the anti-αKlotho sb106 antibody wasgenerated by targeting an extracellular region of αKlotho present inboth the secreted and membrane-anchored forms of αKlotho. Theanti-αKlotho sb106 antibody was obtained from a selection in which apurified complex of mouse αKlotho with human FGFR1c receptor(FGFR1c/αKlotho) was exposed to a synthetic antibody phage-displaylibrary.

In an embodiment, αKlotho polypeptide is used to isolate an antibodythat specifically binds αKlotho polypeptide from the antibody library.In an embodiment, αKlotho complexed with FGFR1c is used to isolate anantibody that specifically binds αKlotho polypeptide from the antibodylibrary.

In another embodiment, the isolated and purified antibody and/or bindingfragment thereof is affinity matured. Affinity maturation can performedas described for the initial selection, with antigen adsorbed to plasticplates, using a for example a phage library comprising variants of theCDR sequences for example as described in Example 8.

A person skilled in the art will appreciate that several methods can beused to produce antibodies and/or binding fragments thereof withspecific binding affinity to folded αKlotho. A method that can be usedis a phage display method. Briefly, a binary αKlotho-FGF1Rc complex isproduced (as described in Example 1) in order to isolate andcharacterize the antibody and/or binding fragment thereof. Phage from ahuman Fab phage-displayed library are selected following several roundsof panning. Phage with specific binding affinity to the binaryαKlotho-FGF1Rc complex, as determined by ELISA, are sequenced and clonedinto vectors designed for production of light chain or heavy chain. Theheavy chain can be for example an IgG, or an IgG isotype such as an IgG1or an IgG4. Antigen binding fragments and IgG polypeptides are thenaffinity purified by using for example Protein A affinity columns.

In another embodiment, a nucleic acid encoding an antibody describedherein is expressed in a host cell to make the antibody and/or bindingfragment thereof. In an embodiment, the method comprises:

-   -   a. expressing in a host cell a nucleic acid encoding an antibody        and/or binding fragment thereof herein disclosed;    -   b. culturing the host cell to produce the antibody and/or        binding fragment thereof; and    -   c. isolating and/or purifying the antibody and/or binding        fragment thereof from the host cell.

In some embodiments, a nucleic acid encoding a single chain antibody isexpressed. In other embodiments, multiple nucleic acids are expressed,for example encoding a nucleic acid encoding an antibody light chain anda nucleic acid encoding an antibody heavy chain.

Suitable host cells and vectors are described above. Vectors and nucleicacids encoding an antibody described herein may be introduced intomammalian cells via conventional techniques such as calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran mediated transfection,lipofectin and other liposome based transfection agents, electroporationor microinjection.

Nucleic acid encoding an antibody described herein may be directlyintroduced into mammalian cells using delivery vehicles such asretroviral vectors, adenoviral vectors and DNA virus vectors.

As described in the Examples, the sb106 antibody was tested in cells andanimal models as follows: the antibody was screened in cultured cellsexpressing native αKlotho or transfected with αKlotho, in rodents (miceand rats), and in human plasma and urine samples from normal individualsand patients with CKD. The procedures tested included immunoblot,immunoprecipitation, immunohistochemistry, immunocytochemistry, andfluorescence-activated cell sorting (FACS).

The ability of the antibody and/or binding fragment herein disclosed toprecipitate soluble αKlotho can be determined using a sequentialimmunoprecipitation-immunoblot assay as described in Example 6.

A further aspect is an assay for detecting αKlotho polypeptide in asample the assay comprising:

-   -   a) contacting a sample with the antibody or binding fragment        described herein under conditions to form an antibody: αKlotho        complex; and    -   b) detecting the antibody:αKlotho complex.

In an embodiment, the assay is for detecting folded αKlotho and theassay is performed under non-denaturing or mildly denaturing conditions.

In an embodiment, the complex is detected directly for example whereinthe antibody is labeled with a detectable tag or fusion moiety. In anembodiment, the complex is detected indirectly using a secondaryantibody specific for the antibody:αKlotho complex.

In an embodiment, the assay is an immunoprecipitation, immunoblot,immunohistochemistry or immunocytochemistry proximity ligation assay(PLA), mass spectroscopy-based techniques and fluorescence-activatedcell sorting (FACS), proximity ligation assay (PLA), and massspectroscopy-based techniques. The antibodies described herein areefficient for immunoprecipitating αKlotho polypeptide and are muchbetter than antibodies such as KM2076 and others [13] tested by theinventors.

In one specific embodiment, the method assay is a sequentialimmunoprecipitation-immunoblot (IP-IB). The IP-IB can be performed forexample as described in the Examples below.

In an embodiment, the method is for detecting soluble αKlotho, forexample wherein the sample is a biological fluid.

A further aspect includes a method for detecting and/or measuringsoluble αKlotho the method comprising:

a) contacting a sample, the sample being a biological fluid, with theantibody or binding fragment described herein under conditions to forman antibody: soluble αKlotho complex; and

-   -   b) detecting and/or measuring the antibody:soluble αKlotho        complex.

Detecting can be performed using methods that are qualitative ormeasured using quantitative methods, for example by comparing to astandard or standard curve.

In an embodiment, the biological fluid sample is blood, or a partthereof such as serum or plasma, or urine.

In an embodiment, the assay is a diagnostic assay. For example, asdetailed in Example 7, the IP-IB assay comprising the antibody and/orbinding fragment thereof herein disclosed is evaluated to test itslinearity and to determine whether it can reliably detect serum αKlotholevels in chronic kidney disease (CKD) patients. As such, pre-determinedamounts of recombinant αKlotho varying from grade 1 CKD to grade 5 CKDalong with serum from a healthy volunteer are used. The results show anincremental relationship between the serum levels and the CKD stage.Further, as shown in FIG. 6B, the IP-IB assay with sb106 shows animportant reduction of urinary αKlotho in patients with CKD.

Yet another aspect relates to a method for screening, for diagnosing orfor detecting kidney insufficiency condition selected from chronickidney disease (CKD) and acute kidney injury (AKI) in a subject, themethod comprising:

-   -   a. measuring the level of αKlotho in a sample from a subject        optionally using an antibody or assay herein disclosed; and    -   b. comparing the level of αKlotho in the sample with a control,        wherein a decreased level of αKlotho in the sample compared to        the control is indicative that the subject has a kidney        condition selected from CKD or AKI.

In an embodiment, the control is a control value derived from a group ofsubjects without CKD or AKI e.g. normal controls.

In an embodiment, the CKD is early CKD, optionally stage 1, stage 2,stage 3, stage 4, stage 5 or stage 6 CKD.

An additional aspect of the disclosure is a method for prognosticatingCKD progression or AKI progression or lack thereof (e.g. recovery orworsening of disease), or extra-renal complication in CKD, which isassessed by measuring the level of αKlotho deficiency.

Accordingly an aspect is a method of prognosticating a likelihood ofrecovery after AKI, the method comprising:

-   -   a. measuring a level of αKlotho in a sample from a subject; and    -   b. comparing the level of αKlotho in the sample with a control,        for example a control value derived from a group of subjects        that did not recover or progressed, wherein an increased level        of αKlotho in the sample compared to the control is indicative        that the subject has an increased likelihood of recovery after        AKI.

In an embodiment, the control is a control value derived from a group ofsubjects that did recover and a decreased level of αKlotho in the samplecompared to the control is indicative that the subject has a decreasedlikelihood of recovery after AKI and/or an increased likelihood ofdisease progression.

Another aspect is a method for prognosticating a likelihood of long termcomplications after AKI, the method comprising:

-   -   a. measuring a level of αKlotho in a sample from a subject; and    -   b. comparing the level of αKlotho in the sample with a control,        for example a control value derived from a group of subjects        with long term complications or with increased number of long        term complications,    -   wherein an increased level of αKlotho in the sample compared to        the control is indicative that the subject has a increased        likelihood of having fewer long term complications after AKI

In an embodiment, wherein the control is a control value derived from agroup of subjects without long term complications or with fewer longterm complications, an decreased level of αKlotho in the sample comparedto the control is indicative that the subject has a increased likelihoodof having long term complications or an increased number of long termcomplications after AKI.

A further aspect is a method for prognosticating the likelihood ofprogression of CKD, the method comprising:

-   -   a. measuring a level of αKlotho in a sample from a subject; and    -   b. comparing the level of αKlotho in the sample with a control,        wherein an increased level of αKlotho in the sample compared to        the control, for example wherein the control is a control value        derived from a group of subjects that or example a control value        derived from a group of subjects that did not recover or        progressed is indicative that the subject has an increased        likelihood of recovering from CKD.

In an embodiment, the control is a control value derived from a group ofsubjects that did recover and a decreased level of αKlotho in the samplecompared to the control is indicative that the subject has a decreasedlikelihood of recovery after CKD and/or an increased likelihood ofdisease progression.

Yet another aspect is a method for prognosticating extra-renalcomplications in CKD, the method comprising:

-   -   a. measuring a level of αKlotho in a sample from a subject; and    -   b. comparing the level of αKlotho in the sample with a control,        wherein an increased level of αKlotho in the sample compared to        the control is indicative that the subject has a higher        likelihood of having fewer extra-renal complications related to        CKD.

In an embodiment, wherein the control is a control value derived from agroup of subjects without extra-renal complications or with fewerextra-renal complications, an decreased level of αKlotho in the samplecompared to the control is indicative that the subject has a increasedlikelihood of having long term complications or an increased number ofextra-renal complications after CKD.

A further aspect is a method for monitoring a subject with a kidneyinsufficiency condition such as CKD or AKI the method comprising:

-   -   a. measuring a level of αKlotho in a sample from a subject; and    -   b. comparing the level of αKlotho in the sample with a previous        reference sample other control,    -   wherein an increased level of αKlotho in the sample compared to        the previous reference sample or other control is indicative        that the subject has an ameliorating kidney condition and a        decreased level of αKlotho in the sample compared to the        previous reference sample or other control is indicative that        the subject has a worsening kidney condition.

The sample can for example be taken after the subject has received atreatment and compared for example to a pre-treatment sample.Alternatively the patient can be monitored after a repeating interval toassess for example if treatment or other intervention is necessary. Inan embodiment, the test is repeated and plotted to assess the subject'sprogression.

In an embodiment, the sample is a biological fluid such as blood, or afraction thereof such as plasma or serum and the method is for exampledetecting soluble αKlotho. In an embodiment the biological fluid isurine.

In another embodiment, the sample is selected from a fresh sample suchas a fresh biological fluid sample or tissue sample (e.g. including notfrozen or one time frozen (e.g.

frozen a single time at the time of obtaining the sample)) and a repeatfrozen sample (e.g. frozen and thawed and frozen biological fluid sampleor repeat frozen tissue sample). In an embodiment, the sample is a fixedsample such as a mildly fixed sample wherein the fixation induceslimited denaturation and/or unfolding.

In an embodiment, the level of αKlotho is measured using an antibody orbinding fragment described herein.

The methods disclosed herein to diagnose, detect or monitor a kidneydisease or prognosticate a kidney disease complication, can be used inaddition to or in combination with traditional diagnostic techniques forkidney disease.

IV. Kits

A further aspect relates to a kit comprising i) an antibody and/orbinding fragment thereof, ii) a nucleic acid, iii) composition or iv)recombinant cell described herein disclosed, comprised in a vial such asa sterile vial or other housing and optionally a reference agent and/orinstructions for use thereof.

In an embodiment, the kit is a diagnostic kit and the instructions aredirected to a method described herein.

The above disclosure generally describes the present application. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theapplication. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

Examples Example 1

A synthetic antibody library was screened and an antigen-bindingfragment (Fab) with high affinity for human and rodent αKlotho wasgenerated. This novel antibody, sb106, was characterized usingrecombinant proteins, cultured cells, and body fluids and tissues fromhumans and rodents. αKlotho levels in serum and urine in human androdents can be accurately quantified, and it is demonstrated that bothserum and urine αKlotho are dramatically reduced in early human CKD. Thesb106 antibody is specific to the α-form of Klotho and is the firstknown one to successfully pull down αKlotho from patient serum samples,in a clean and specific manner, as compared to currently commerciallyavailable αKlotho detection reagents. In cells, it can immunoprecipitateαKlotho and label it by immunocytochemistry. In animals, the antibody isefficient at immunoprecipitating αKlotho from plasma. The ability of thesb106 antibody to detect small quantities of αKlotho from biologicalfluids makes it a valuable reagent for diagnosis of diseases where thelevel of αKlotho is abnormal. Moreover, sb106 antibody is valuable as aresearch reagent in studies of physiologic and pathologic states thatinvolve any FGF23-mediated signaling pathways. It can be used inspecific assays for soluble αKlotho in human and rodent samples such asserum using a variety of techniques such as enzyme-linked immunosorbentassay (ELISA), proximity ligation assay (PLA), and massspectroscopy-based techniques.

Example 2 Preparation of the Binary αKlotho-FGFR1c Complex

The ligand-binding domain of human FGFR1c (D142 to R365) was expressedin E. coli, refolded in vitro from inclusion bodies, and purified bypublished methods [72,73]. The extracellular domain of murine αKlotho(A35 to K982) was expressed in HEK293 cells with a C-terminal FLAG tag,and the binary complex of the αKlotho ectodomain and the FGFR1cligand-binding domain was prepared as described [9].

Isolation and Characterization of Sb106

Sb106 was isolated from a synthetic human Fab phage-displayed library(Library F)[74]. Binding selections, phage ELISAs and Fab proteinpurification were performed as described[67,75,76]. Briefly, phage fromlibrary F were cycled through rounds of panning with the binary complexof αKlotho extracellular domain and FGFR1c ligand-binding domain on96-well Maxisorp Immunoplates (Fisher Scientific, Nepean, ON, Canada) asthe capture target. After 5 rounds of selection, phage were producedfrom individual clones grown in a 96-well format and phage ELISAs wereperformed to detect specific binding clones. Clones that showed bindingwere subjected to DNA sequencing. A competitive binding ELISA wasperformed by pre-incubating sb106-phage with serial dilutions of solublehuman αKlotho (50-0.0005 nM×1 hour) prior to binding to an ELISA platecoated with human αKlotho. The genes encoding for variable heavy andlight chain domains of sb106 were cloned into vectors designed forproduction of light chain or IgG1 heavy chain, respectively, andsb106-IgG was expressed from 293F cells (Invivogen, San Diego, Calif.USA). Fab and IgG proteins were affinity-purified on Protein A affinitycolumns (GE Healthcare, Mississauga, ON, Canada).

Purification of Proteins

The binary complex of FGFR1c ligand-binding domain and murine αKlothoectodomain (referred to as αKlotho-FGFR1c complex) was prepared by apublished protocol[9].

The N-terminally hexahistidine tagged, mature form of human FGF23 (Y25to I251) was expressed in E. coli and purified by published protocols[73,74,77].

Analysis of Fab Binding to αKlotho-FGFR1c Complex by SPR Spectroscopy

Real time protein-protein interactions were measured using a Biacore2000 surface plasmon resonance (SPR) spectrometer (Biacore AB/GEHealthcare) at 25° C. in HBS-EP buffer (10 mM HEPES-NaOH, pH 7.4, 150 mMNaCl, 3 mM EDTA, 0.005% (v/v) polysorbate 20). Proteins were covalentlycoupled through their free amino groups to carboxymethyl (CM) dextran ofresearch grade CM5 biosensor chips (Biacore AB/GE Healthcare). Proteinswere injected over a biosensor chip at a flow rate of 50 μl min⁻¹, andat the end of each protein injection (180 s), HBS-EP buffer (50 μlmin⁻¹) was flowed over the chip to monitor dissociation for 180 s.Injecting 2.0 M NaCl in 10 mM sodium acetate, pH 4.5, or 10 mMsodium/potassium phosphate, pH 6.5 regenerated the chip surface inbetween protein injections. The data were processed with BiaEvaluationsoftware version 4.1 (Biacore AB/GE Healthcare). For each proteininjection, the non-specific SPR responses recorded for the control flowchannel were subtracted from the responses recorded for the sample flowchannel.

To examine whether Fabs selected by ELISA bind to the αKlotho-FGFR1ccomplex, the binary receptor complex was immobilized on a CM5 chip (˜42fmol mm⁻² of chip flow channel). To control for non-specific binding,bovine β-glucuronidase (Sigma-Aldrich), which is structurally related toeach of the two extracellular glycosidase-like domains of αKlotho, wascoupled to the control flow channel of the chip (˜45 fmol mm⁻² of flowchannel). 100 nM of each Fab were injected over the chip. As a control,binding of FGF23 to the immobilized αKlotho-FGFR1c complex was examined.

To test if the Fabs can compete with FGF23 and/or binding to theαKlotho-FGFR1c complex, FGF23 was immobilized on a CM5 chip (˜16 fmolmm⁻² of chip flow channel). FHF1B, which is structurally similar to FGFsbut has no FGFR binding[77], was used as control for non-specificbinding (˜15 fmol mm⁻² of control flow channel). 100 nM of Fab mixedwith 10 nM of αKlotho-FGFR1c complex (HBS-EP buffer) was injected overthe chip. For control, the binding competition was carried out withFGF23 as the competitor in solution.

Cell Culture, Animal and Human Studies

Cell lines: normal rat kidney (NRK) cells with native αKlotho expression(ATCC, Manassas, Va., USA), and HEK293 cells transfected with vectoronly, full-length transmembrane murine αKlotho, extracellular domain ofmurine αKlotho with C-terminal FLAG tag, or full-length murineβKlotho[78]. Cells were cultured at 37° C. in a 95% air, 5% CO₂atmosphere, passed in high-glucose (450 mg/dl) DMEM supplemented with10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100mg/ml).

Animal studies were approved by the University of Texas SouthwesternMedical Center Institutional Animal Care and Care Committee. All animalswere housed in the Animal Resource Center and experiments were performedin fully approved laboratories. Species used include: Sprague-Dawleyrats (Harlan. Indianapolis, Ind.), Klotho transgenic overexpressors(Tg-Klotho; EFmKL46 line)[79], homozygous αKlotho hypomorphic mice(KI/KI)[80], and their wild type littermates (129sv background).

Clinical history and routine lab data were obtained from electroniccharts. Blood samples from antecubital venipuncture were spun, and theserum was frozen at −80° C. Fresh urine was spun at 4,000 g and thesupernatant was frozen at −80° C.

Immunocytochemistry and Immunohistochemistry

HEK293 cells transfected with vector or the stated αKlotho plasmids andseeded (1.8×10⁵ cells/ml) on 12-well glass cover slips pre-treated withpoly-lysine and grown overnight. The cells were washed (4° C. PBS×3),fixed with 3% paraformaldehyde (4° C.×10 min), washed (ice cold PBS×3),blocked with 1% BSA (PBS 44° C.×10 min), incubated with sb106-Fab (5ug/ml in 1% BSA, PBS) washed (PBS 4° C.×5), incubated withanti-FLAG-Alexa488 (Cell Signaling; diluted 1:400 in PBS containing 1%BSA; 1 hour; 20° C.), washed (PBS; 4° C.×4), and then inverted ontoglass slides containing a drop of antifade with DAPI (Invitrogen) anddried at room temperature in the dark. After 24 hours, the slides werestored at −20° C. Images were obtained on a WaveFX spinning discconfocal microscope.

The parathyroid and thyroid (en bloc with the trachea) were from adultSprague Dawley rats. For non-fixed fresh parathyroids, tissues wereembedded in OCT medium and frozen with isopentane pre-cooled in liquidN2 immediately. For fixed parathyroid samples, tissue was immersed in 4%paraformaldehyde in PBS pH 7.4 at 4° C. overnight, washed with PBS, andembedded in OCT medium and frozen with isopentane pre-cooled in liquidN2. Four μm thick cryostat sections were made, washed in PBS (15 min),and permeabilized in 0.1% TritonX-100 (10 min). For labeling, sectionswere blocked (PBS, 1.5% BSA, 10% goat serum; 40 min) and incubated withthe primary antibody sb106 (21 pg/ml in blocking solution; 4° C.overnight). After washing (PBS), sections were incubated with the Alexa546 goat anti-human IgG (1:800 dilution, Invitrogen) for 1 hour at roomtemperature. After additional washes with PBS, the sections were fixedwith 4% paraformaldehyde in PBS, washed with PBS, and mounted andvisualized with a Zeiss LSM510 microscope.

Example 3: αKlotho Assays and Detailed Methods

The ELISA was performed as instructed by the manufacturer(Immuno-Biological Laboratory, Japan). For the IP-IB assay, typically 50μl of serum or urine were diluted with KRH buffer [25 mM Hepes-NaOH (pH7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.3 mM CaCl₂), 1.3 mM KH2PO4]to a final volume of 0.5 ml incubated with 2 pg of sb106-Fab overnightat 4° C. in low binding, siliconized tubes. Sepharose beads (50 pL)conjugated with anti-FLAG antibody (50% v/v) prewashed 3× with KRHbuffer were added, incubated (4° C.×2 h), and washed (KRH-500 μl pertube×3; 22° C.). The immune complex was eluted with 2×SDS sample loadingbuffer (50 μl; 100° C.×3 min; 4° C.×3 min; spun), and fractionated bySDS-PAGE, transferred to nitrocellulose membranes and blotted withanti-KL1 antibody (KM2076, 1:4000 or 3.1 mg/mL, 1:10000 dilution) anddiluent (Dako#S3022, Carpinteria, Calif., USA) overnight (4° C.,rocker). The membrane was washed (×3, Tris-buffered saline with 0.1%Tween; TBS-T), exposed to anti-rat IgG2A (LSBio cat#LS-059051, 1:20000in 5% milk/2% goat serum/TBS-T×1 h) and washed (×3 TBS-T). Forchemiluminescence, the membrane was covered with SuperSignal West FemtoMaximum Sensitivity substrate (Thermo Scientific, Rockford, Ill., USA)and exposed for 30-90 s. The 130-kD bands were scanned, and density wascompared with internal control samples of know amount of Klotho usingAdobe Photoshop CS4.

Example 4: Identification of an Anti-αKlotho Synthetic Fab

After rounds of biopanning of a phage-displayed synthetic Fab library onrecombinant αKlotho ectodomain complexed with the ligand-binding domainof fibroblast growth factor receptor (FGFR)1c, several binding phageswere identified. Clone sb106 (FIG. 1A) was chosen for furthercharacterization. In phage ELISA (FIG. 1B), sb106-phage bound to bothhuman and mouse αKlotho, demonstrating cross species reactivity, and toeither αKlotho alone or in complex with FGFR1c, indicating that itsepitope is not obscured by co-receptor complex formation. Sb106-phagedid not bind to FGFR1c alone, neutravidin (NAV) or bovine serum albumin(BSA). Sb106 binds to human αKlotho with affinity in the single-digitnanomolar range (IC50=1.7 nM, FIG. 1C). Sb106-Fab also binds with highaffinity to the binary αKlotho-FGFR1c complex immobilized on a biosensorchip (FIG. 7A) and it does not interfere with ternary complex formationbetween FGF23, αKlotho and FGFR1c (FIG. 7B).

Example 5: Characterization of the Anti-αKlotho Fab Sb106

Using the unique CDR sequences of sb106 (FIG. 1A), both Fab andfull-length IgG proteins were produced (e.g. Fab was produced inbacterial cells and IgG proteins in 293F cells). Sb106 was highlyreactive against αKlotho under native conditions. Immunoblot signalsunder denaturing conditions against mouse, rat, and human kidney tissuewere weak, but in samples from transgenic mice overexpressingαKlotho[79], sb106-Fab detected a band corresponding to the full-lengthextracellular domain of αKlotho (FIG. 2A). In cultured cells, sb106-Fabwas not able to detect αKlotho in immunoblots under denaturingconditions with lysates from NRK cells expressing native αKlotho but itwas able to detect overexpressed antigen in cell lysates from HEK293cells transfected with αKlotho (FIG. 2B). In immunohistochemistry withfreshly frozen unfixed rat parathyroid tissue (and other tissues knownto express αKlotho, sb106-IgG detected αKlotho but the same tissue wasnegative when fixed (FIG. 2C), suggesting that sb106 binds to thenatively folded FGFR1-αKlotho complex (FIG. 1B). In immunocytochemicalstains with freshly fixed cells, unequivocal staining was obtained inHEK293 cells heterologously overexpressing αKlotho but not in cellsoverexpressing βKlotho (FIG. 2D). The cells were seeded at 1.8×10⁵/ml on12-well glass cover slips treated with poly-lysine and grown overnight.Cells were washed 3 times with ice cold PBS, fixed for 10 min on ice (3%paraformaldehyde), washed 3 times with cold PBS, blocked for 10 min (1%BSA in cold PBS). Cells were then incubated with sb106-Fab (5 pg/ml in1% BSA in PBS) for 1 hr. Cells were washed 5 times with cold PBS (2 mineach) and then incubated with anti-FLAG-Alexa488 (the C-terminus of theFab light chain contains a Flag epitope tag) (1:400 in 1% BSA in PBS)for 1 hr, while being protected from light. Cells were washed 4 timeswith cold PBS (5 min each). The glass coverslips were then inverted ontoglass slides containing a drop of antifade reagent with DAPI. Imageswere collected on a spinning disc confocal microscope. Even in cellsoverexpressing αKlotho, prolonged fixation greatly diminished orabolished the staining with sb106. These data indicate that sb106 reactsspecifically with natively folded human, rat, and mouse αKlotho but notwith denatured αKlotho protein.

Example 6: Immunoprecipitation of αKlotho

The ability of sb106-Fab to precipitate soluble αKlotho was tested usinga sequential immunoprecipitation-immunoblot (IP-IB) assay. Sb106-Fabpulled down αKlotho from total cell lysates and conditioned cell culturemedium and from αKlotho-overexpressing cells (FIG. 3A). The sb106-Fabpull-down was compared with that of an anti-FLAG antibody using solubleαKlotho with a C-terminal FLAG tag in HEK293 cells. Sb106-Fab andanti-FLAG precipitated proteins with the exact same electrophoreticmobilities.

Sb106-Fab precipitated a ˜130 kDa protein from human, mouse, and ratsera that reacted with the anti-αKlotho antibody KM2076 (FIG. 3B). IPfrom urine also showed a ˜130 kDa band (FIG. 3B), whereas the post-IPurine samples did not show such a band in immunoblot. To further supportthe authenticity of the IP-IB band by sb106, the intensity of this bandwas examined in human sera from a normal individual vs. a patient withCKD stage 5, and sera from a wild type mouse vs. a homozygous αKlothohypomorph (FIG. 3C). Only the −130 kDa band (FIG. 3C) was reduced inhuman advanced CKD and was absent in the αKlotho-deficient mice(kl/kl)[80].

Example 7: αKlotho Levels in Human CKD

The IP-IB method was tested to determine whether it can reliablydetermine serum αKlotho levels from a single center database of CKDpatients. Recombinant human αKlotho was spiked in known amounts to testthe linearity of the assay as well as the extrapolated y-intercept.IP-IB was performed with sera from a normal healthy volunteer and apatient with stage 5 CKD spiked with a range of different concentrationsof recombinant αKlotho (FIG. 4A). There was graded increase in signalwith the incrementally inoculated exogenous αKlotho. The serum from theCKD patient also showed increases in signal with increasing exogenousαKlotho but, at any given concentration of αKlotho, the signal intensitywas lower than the normal serum.

Interestingly, the serum from the healthy volunteer gave the same signalin the absence or presence of a protease inhibitor cocktail, whereas theserum from the CKD patient displayed a marked increase in measuredαKlotho levels in the presence of a protease inhibitor (FIG. 4B). Thesefindings suggest that while endogenous αKlotho exists in a stable steadystate in uremia, the exogenously added αKlotho may undergo proteolysisin uremic serum but not in normal serum. A quantitative summary of thespiking experiment is shown in FIG. 4C. Both healthy and CKD sera showedlinear responses to αKlotho inoculation but the signal from CKD sera hasa lower slope. When protease inhibitors were included, the slope of theCKD line approached that of the healthy subject without affecting itsintercept. Extrapolation to zero inoculation showed that the serum fromthe normal individual had 31.1 μM αKlotho while that from the CKDpatient had 8.5 μM αKlotho. Similar extrapolations were obtained from anumber of other subjects with normal renal function or with CKD.

The nature of patients recruited from the CKD clinic resembles thenational profile of CKD where diabetes and hypertension predominate(Table 1). Despite the scatter, there is a clear progressive decline ofαKlotho with stages of CKD (FIG. 5A). The decrease in serum αKlothooccurred early in CKD, and preceded high FGF23, high PTH, andhyperphosphatemia (Table 1). The IP-IB assay was directly compared witha commercial αKlotho ELISA kit with the same samples (FIG. 5B). Overall,there is a correlation between the two but there is separation on bothsides of the line of identity. In fresh samples, the ELISA shows highervalues than IP-IB (grey diamonds to the left of the line of identity,FIG. 5B) but in samples that have been through one or more cycles offreeze-thaw, the ELISA values are much lower (black diamonds to theright of the line of identity, FIG. 5B). When the exact same sampleswere tested by the two methods before and after repeated freeze-thaw,the IP-IB assay gave more stable results while the ELISA values droppedrapidly (FIG. 5C).

Low urinary αKlotho in human CKD patients by directly immunoblottingurine was previously described[12]. The IP-IB assay with sb106-Fabshowed dramatic reduction of urinary αKlotho in CKD patients (FIG. 6A).In contradistinction from serum, the ELISA yielded more comparablevalues to the IP-IB assay in the urine but the magnitude of decrease inαKlotho concentration is more dramatic when detected by the IP-IB assaythan by ELISA (FIG. 6B). These results show that human CKD is a state ofαKlotho deficiency in both serum and urine.

Hence using an immunoprecipitation-immunoblot (IP-IB) assay, both theserum and urinary levels of full-length soluble αKlotho was measured inhumans and it was established that human CKD is associated with αKlothodeficiency in serum and urine. αKlotho levels were detectably lower inearly CKD preceding disturbances in other parameters of mineralmetabolism and levels progressively declined with CKD stages.Exogenously added αKlotho is inherently unstable in the CKD milieu.

Antibody-based reagents are valuable tools in both research and clinicalsettings for detection of proteins, protein isolation and purification,and numerous downstream applications. The commercial reagents availablefor αKlotho detection are limiting; for example there are no antibodiesfor specifically detecting natively folded αKlotho protein. Moreover,the commercial ELISA kit for αKlotho detection yields highly variableresults.

Synthetic antibodies with designed antigen-binding sites can befine-tuned and tailored for molecular recognition of vast repertoires oftargets. Coupled with in vitro phage-display, selections are performedin the absence of tolerance mechanisms that eliminate self-reactiveantibodies. Selections with an antibody library yielded sb106, anantibody with specificity for natively folded human, mouse and ratαKlotho.

In addition to its role in mineral metabolism, soluble αKlothocirculates in many bodily fluids and has multiple “house-keeping”functions that maintain cellular integrity throughout the body. Althoughthe mechanism of action of soluble αKlotho remains poorly understood,the biologic impact of αKlotho deficiency is unequivocally shown[81].αKlotho transcripts are present in multiple organs but the kidney by farhas the highest expression[80]. CKD is a state of multiple metabolicderangements and is a complex syndrome from accumulation ofunder-excreted endogenous and exogenous toxins as well as deficiency insubstances responsible for health maintenance.

There is evidence in experimental animals that both AKI and CKD arestates of systemic αKlotho deficiency. Not only is this an early andsensitive biomarker, restoration of αKlotho can ameliorate the renaldysfunction. Independent from its renoprotective effects, αKlotho canalso reduce the extrarenal complications in CKD[12,82]. Based on thepreclinical data, anti-αKlotho antibody may have both diagnostic andprognostic value.

Validation of the IP-IB Assay and Comparison with the Commercial ELISA

Available commercial assays for αKlotho have no consistent correlationbetween them[46, 83]. Studies in healthy humans and CKD patients basedon one ELISA[58] have yielded contradictory results. The absolute levelsof αKlotho in normal and CKD ranged from 0.4[47] to over 2000 pg/ml[41]with most readings in the mid to high hundreds[48, 50, 55, 58-60,83].Based on this assay, αKlotho levels have been described to be low[48,52, 54, 57-60], no relationship to[40, 41, 50, 51, 53] or evenincreased[44, 47] with decreasing glomerular filtration rate (GFR).Likewise, αKlotho levels have been reported as not changed or decreasingwith age[42, 53, 58, 59]. This renders the interpretation of humanαKlotho data nearly impossible, and the collective data derived fromdifferent centers will have no value.

A high affinity synthetic antibody that recognizes αKlotho in itsnatively folded conformation (FIGS. 1-3) was generated. Sb106-Fab or IgGpulls down αKlotho from cell lysate, culture medium, serum, and urine.Additional bands may be shorter fragments of αKlotho but the intensityof these bands did not decrease in the kl/kl mice arguing against thispossibility. The −130 kDa band has been analyzed which is full lengthsoluble αKlotho; something that the ELISA cannot achieve.

The linearity of the spiking experiment indicates that all theinoculated αKlotho is detected (FIG. 4). An unexpected finding was thatexogenously added recombinant αKlotho is proteolytically degraded inuremic serum whereas no such phenomenon was observed in normal sera.This challenges the view that the low αKlotho in kidney disease stemssolely from decreased production and opens up additional mechanisms andnew avenues for investigation. In addition to uncovering new mechanismsof αKlotho deficiency in CKD, this may have significant implications interms of recombinant αKlotho replacement.

There is graded reduction in serum αKlotho with advancing CKD (FIG. 5A).The coefficient of variation of the IP-IB assay was 4% for serum and 7%for urine. The IP-IB assay also showed very low urinary αKlotho inadvanced CKD (FIG. 6). In fact, the reduction in urinary αKlotho is moredramatic than that in serum and may represent a more sensitive markerfor CKD.

Both IP-IB and the commercial ELISA detected the low urine αKlotho inCKD, although the absolute levels of αKlotho are much higher with theELISA assay and the percent reduction is not the same as with the IP-IBassay. With drastic reduction in urinary αKlotho levels in CKD, the twoassays yielded the same conclusion with quantitative differences. Thesituation in serum is different. Although there is overall positivecorrelation, the comparison of the two assays completely segregated intotwo groups (FIG. 5B). The fresh samples showed higher readings for theELISA while the stored samples yielded extremely low results with theELISA. One possibility is that the ELISA is measuring αKlotho and someother reacting proteins in fresh samples. While the IP-IB assay did losesome efficacy with repeated freeze-thaw, this is a much more seriousproblem with the ELISA.

Another advantage of the IP-IB assay is that it can measure αKlotho inboth humans and rodents equally well, whereas the use of the currentlyavailable ELISA in rodent can potentially be problematic as it detectsvery high circulating αKlotho levels in rats with CKD which is a stateof pan-αKlotho deficiency.[68]

Example 8

Additional CDR sequences are provided in Table 2. Homologous mutationswere introduced at each amino acid position, meaning that for eachposition either the original amino acid was retained or the closest“homolog” to that amino acid (e.g. conservative amino acid change) wasintroduced and a new Fab-phage library was constructed. Selections wereperformed using the new library using the alphaKlotho-FGFR1c complex asan antigen. Clones that bound to the antigen were isolated and sequencedand are shown in Table 2. The binding affinity is expected to be similaror better than Sb106.

Example 9 Human Studies

Nine human subjects (49.066.2 years) who underwent right heartcatheterization were enrolled for this study. During right heartcatheterization, suprarenal and infrarenal vena caval blood samples wereobtained and sera were immediately separated after centrifugation at 4°C. and stored at −80° C. for future study. Serum αKlotho was determinedby immunoprecipitation-immunoblot assay described herein. Briefly, 0.1ml serum was immunoprecipitated with a synthetic anti-αKlotho Fab(sb106) and immune complex was eluted with Laemmli sample buffer, andsubject to immunoblot with KM2076 antibody. The specific signals on theautoradiograms based on 130 kD mobility were quantified with ImageJProgram (National Institutes of Health (NIH), Bethesda, Md.).

Animal Studies

αKlotho hypomorphic (kl/kl) mice, kl/kl mice and their wild-type (VVT)littermates were maintained at the Animal Research Center at theUniversity of Texas Southwestern Medical Center. Currently all mice are129 S1/SVImJ (129 SV) background age from 6 to 8 weeks. NormalSprague-Dawley (SD) rats (220-250 g body weight) were purchased fromCharles River Laboratories (Wilmington, Mass.). For αKlotho clearancestudy, rats underwent bilateral nephrectomy (anephric rats) orlaparotomy with manual manipulation of the kidneys (sham rats). Rats ormice were intravenously or intraperitoneally injected once with labeledfull extracellular domain of recombinant mouse αKlotho protein (rMKI)(R&D Systems, Minneapolis, Minn.) at a dose of 0.1 mg/kg body weight. Toexamine if secretases modulate blood αKlotho, doxycline hyclate(Sigma-Aldrich, St. Louis, Mo.), an α-secretase inhibitor at 25mg/kg/day, and/or β-secretase inhibitor III (Calbiochem, Billerica,Mass.) at 2.5 mg/kg/day were intraperitoneally injected into normal VVTmice daily for 2 days, blood and kidneys were harvested at 48 hours todetermine serum and renal αKlotho.

Antibodies

Rat monoclonal anti-human Klotho antibody, KM20761,2 was used forimmunoblotting and immunoelectron microscopy; and the syntheticanti-αKlotho antibody sb10663 was used for immunoprecipitation of serumKlotho.

Clearance of Labeled αKlotho in Rats and Mice

Normal Munich Wistar rats (220-250 g BW) were anesthetized with Inactin(100 mg/kg BW), and a bonus of labeled αKlotho was injected through thejugular vein (0.1 mg/kg BW). For the experiment of injection of125I-labeled αKlotho or 125I-labeled albumin, fluid collection byfree-flow micropuncture of Bowman's space, and proximal convolutedtubules was performed using our published methods. In brief, the leftkidney was exposed, and the left ureter was catheterized for urinecollection. Proximal tubules were identified by their characteristicconfiguration after lissamine green dye injection and punctured withglass capillaries. The volume of fluid was measured in a calibratedconstant-bore glass capillary. The radioactivity of fluids wasdetermined by scintillation accounting and normalized to fluid volume.At specified time points, blood was drawn from retro-orbital venoussinus, and spot urine was collected. 125I-labeled αKlotho or125I-labeled albumin in collected urine and serum was quantified byscintillation counting. Homogenates of different organs were made andradioactivity in organ homogenates was measured by scintillationcounting, and normalized to protein in organ homogenates. Organ sections(10 mm) were subjected to autoradiography

Immunoelectron Microscopy

Mouse recombinant Klotho protein (0.1 mg/kg BW) was intraperitoneallyinjected once into kl/kl mice and mice were sacrificed 24 hours afterinjection. Kidneys were harvested and fixed with 2.5% paraformaldehydevia aortic perfusion, removed, and post-fixed in 4% paraformaldehyde (4°C. for 4 hours). Immunogold labeling of ultrathin frozen tissue sectionswas performed as described.21 Kidney cortex was infiltrated with 2.3 Msucrose overnight, frozen in liquid nitrogen, and 70-80-nm-thicksections were made (Ultramicrotome Reichert Ultracut E; LeicaMicrosystems, Wetzlar, Germany) and mounted on Formvar-coated nickelgrids. The sections were incubated with KM2076 antibody and followed byincubation with gold conjugated protein A (10-nm gold particles,Sigma-Aldrich) for 60 minutes. After staining with uranyl acetate,sections were visualized with Jeol 1200 EX transmission electronmicroscope (Jeol Ltd., Akishima, Japan).

Results

The role of the kidney in circulating αKlotho production and handlingwas examined. Serum levels of αKlotho protein in suprarenal andinfrarenal vena cava of normal rats by direct puncture and humansubjects who underwent right heart catherization. All patients hadeGFR≥60 ml/min/1.73 m². Similar infrarenal-to-suprarenal increment incaval αKlotho level was observed in both rat and human serum samples.Serum αKlotho levels were plotted against serum erythropoietin, awell-known renal-derived hormone, and it was found that as serumerythropoietin rose, and serum creatinine (SCr) decreased frominfrarenal-to-suprarenal inferior vena cava, whereas αKlotho increasedindicating that the kidney secretes αKlotho into the circulation.

When both kidneys were removed from rats, serum αKlotho level droppedsignificantly to about half the normal level in one day. The anephricstate did not permit studies to continue for longer than 40-50 hours.

The method of αKlotho clearance from circulation was investigated. Thelevels of circulating exogenous αKlotho protein in anephric rats weresimilar to those in normal rats immediately after injection, but thehalf-life of exogenous αKlotho protein in normal rats was much shorterthan that in anephric rats and the half-life of endogenous αKlotho uponnephrectomy closely approximates that of exogenous αKlotho in theanephric rats. Further experiments examining the anatomic fate ofintravenous injected exogenous labeled αKlotho supported that the kidneymay be a major organ of αKlotho uptake as well as its excretion

Injected labeled αKlotho protein was prominently distributed in thekidney and spleen, sparsely in the heart, and not detectable in aorta,brain, and muscle. Further experiments tracking clearance ofradioactively labelled exogenous αKlotho in serum and urine, supportedthat αKlotho protein is cleared from blood through the kidney to theurine.

Based on these and further experiments, it was determined that the (1)the kidney produces and releases soluble αKlotho into the systemiccirculation by secretases-mediated shedding of the ectodomain ofαKlotho, (2) the kidney is an important organ to clear soluble αKlothofrom the circulation, (3) αKlotho traffics across renal tubules frombasolateral to intracellular location and is then secreted across theapical membrane into the urinary lumen.

TABLE 1 Characteristics of human subjects Serum Serum Serum 25(OH)Gender PCr Pi HCO₃ ⁻ PTH FGF23 Vitamin D Etiology of CKD Subject n Age(M/F) (mg/dl) (mg/dl) (mM) (pg/ml) (pg/ml) (ng/ml) (number subjects*)Healthy 34 50 ± 17 14/20  0.8 ± 0.2 3.6 ± 0.6 23 ± 2  59 ± 25 30 ± 10 32± 10 None CKD1 10 43 ± 10  7/3  0.8 ± 0.1 3.9 ± 0.5 25 ± 2  47 ± 19 61 ±23 26 ± 7 DM (1) HTN (3) GN (7) CKD2 11 50 ± 22  4/7  1.1 ± 0.2 3.6 ±0.5 26 ± 2  56 ± 22 70 ± 27 21 ± 13 DM (2), HTN (4), GN (4), RK (3) CKD310 57 ± 17  5/5  1.7 ± 0.4^(#) 3.2 ± 0.8 25 ± 3  86 ± 51 79 ± 18^(#) 25± 8 DM (3), HTN (7), GN (3), IN (1) CKD4 14 62 ± 13  8/6  2.7 ± 0.6^(#)3.5 ± 0.9 24 ± 3 202 ± 101^(#) 204 ± 173^(#) 21 ± 8 DM (4), HTN (10), GN(3), RK (1) CKD5 11 62 ± 12  5/6  4.7 ± 2.0^(#) 5.1 ± 3.5^(#) 21 ± 3 223± 188^(#) 580 ± 427^(#) 21 ± 9^(#) DM (7), HTN (7), GN (2) Dialysis 1450 ± 12  6/8 11.9 ± 15.6^(#) 4.8 ± 1.7^(#) 22 ± 5 500 ± 650^(#) 760 ±286^(#) 26 ± 8 DM (7), HTN (10), GN (5), PKD (1) n = number of subjects;PCr = plasma creatinien; GFR = estimated glomerular filtration rate;Serum Pi = serum phosphorus; Serum HCO3⁻ = serum bicarbonate PTH =parathyroid hormone; FGF23+ Fibroblast growth factor 23; DM = Diabetesmellitus; HTN = hypertension; GN = glomerulonephrititis; RK = Remnantkidney; IN = Interstitial nephritis; PKO = Polycystic kidney disease.*Some patients carry more than one diagnosis. Results are shown as mean± standard deviation ^(#)p < 0.05 compared to healthy volunteers. ANOVA

TABLE 2 CDR sequence variations

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Specifically, the sequences associated with eachaccession numbers provided herein including for example accessionnumbers and/or biomarker sequences (e.g. protein and/or nucleic acid)provided in the Tables or elsewhere, are incorporated by reference inits entirely.

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1. An antibody and/or binding fragment thereof, wherein the antibodyand/or binding fragment thereof specifically binds to an epitope of aαKlotho polypeptide with a dissociation constant (K_(D)) of about 10 nMor less, as measured by competitive ELISA assay.
 2. The antibody and/orbinding fragment thereof of claim 1 wherein the αKlotho polypeptide is afolded αKlotho polypeptide.
 3. The antibody and/or binding fragmentthereof of claim 1 or 2, wherein the antibody and/or binding fragmentthere specifically binds to an epitope of a αKlotho polypeptide with adissociation constant (K_(D)) of about 2 nM or less, as measured bycompetitive ELISA assay.
 4. The antibody and/or binding fragment thereofof any one of claims 1 to 3, wherein the antibody or binding fragmentthereof comprises a light chain variable region and a heavy chainvariable region, the light chain variable region comprisingcomplementarity determining region CDR-L3 and the heavy chain variableregion comprising complementarity determining regions CDR-H1, CDR-H2 andCDR-H3, with the amino acid sequences of said CDRs comprising one ormore of the sequences set forth below: a. CDR-L3; (SEQ ID NO: 1)X₁X₂X₃X₄PX₅,

wherein X₁ is A or S, X₂ is G or A, X₃ is Y or F, X₄ is S or A, X₅ is Ior V; b. CDR-H1: (SEQ ID NO: 2) X₆X₇X₈X₉X₁₀X₁₁,

wherein X₆ is I or V, X₇ is S or A, X₈ is Y, F or S, X₉ is Y, F or S,X₁₀ is S or A and is I or V; c. CDR-H2; (SEQ ID NO: 3)X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁,

wherein and X₁₂ is Y, F or S, X2₁₃ is I or V, X₁₄ is S or A, X₁₅ is P orS, X₁₆ is S or A, X₁₇ is Y or F, X₁₈ is G or A, X₁₉ is Y or F, X₂₀ is Tor S and X₂₁ is S, A or Y; and/or d. CDR-H3: (SEQ ID NO: 4)X₂₂X₂₃VYX₂₄X₂₅X₂₆X₂₇WX₂₈GX₂₉GM,

wherein X₂₂ is Y or F, X₂₃ is Y or F, X₂₄ is A or S, X₂₅ is S or A, X₂₆is H or N, X₂₇ is G or A, X₂₈ is A or S and X₂₉ is Y or F.
 5. Anantibody and/or binding fragment thereof comprising a light chainvariable region and a heavy chain variable region, the light chainvariable region comprising complementarity determining region CDR-L3 andthe heavy chain variable region comprising complementarity determiningregions CDR-H1, CDR-H2 and CDR-H3, with the amino acid sequences of saidCDRs comprising one or more of the sequences set forth below: a. CDR-L3;(SEQ ID NO: 1) X₁X₂X₃X₄PX₅,

wherein X₁ is A or S, X₂ is G or A, X₃ is Y or F, X₄ is S or A, X₅ is Ior V; b. CDR-H1: (SEQ ID NO: 2) X₆X₇X₈X₉X₁₀X₁₁,

wherein X₆ is I or V, X₇ is S or A, X₈ is Y, F or S, X₉ is Y, F or S,X₁₀ is S or A and X₁₁ is I or V; c. CDR-H2; (SEQ ID NO: 3)X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁,

wherein and X₁₂ is Y, F or S, X2₁₃ is I or V, X₁₄ is S or A, X₁₅ is P orS, X₁₆ is S or A, X₁₇ is Y or F, X₁₈ is G or A, X₁₉ is Y or F, X₂₀ is Tor S and X₂₁ is S, A or Y; and/or d. CDR-H3: (SEQ ID NO: 4)X₂₂X₂₃VYX₂₄X₂₅X₂₆X₂₇WX₂₈GX₂₉GM,

wherein X₂₂ is Y or F, X₂₃ is Y or F, X₂₄ is A or S, X₂₅ is S or A, X₂₆is H or N, X₂₇ is G or A, X₂₈ is A or S and X₂₉ is Y or F.
 6. Theantibody and/or binding fragment thereof of claim 4 or 5, wherein thecomplementarity determining regions comprise the amino acid sequencesselected from SEQ ID NOs: 5-120, optionally as set forth below: Lightchain variable region: CDR-L3: (SEQ ID NO: 5) AGYSPI;

Heavy chain variable region: CDR-H1: (SEQ ID NO: 6) ISYYSI; CDR-H2: (SEQID NO: 7) YISPSYGYTS; and/or CDR-H3: (SEQ ID NO: 8) YYVYASHGWAGYGM.


7. The antibody and/or binding fragment thereof of claim any one ofclaims 4 to 6, wherein the light chain variable region further comprisescomplementarity determining regions CDR-L1 and/or CDR-L2 comprising theamino acid sequences set forth below: CDR-L1: (SEQ ID NO: 9) QSVSSAand/or CDR-L2: (SEQ ID NO: 10) SAS.


8. The antibody and/or binding fragment thereof according to any one ofclaims 1 to 7, wherein the antibody and/or binding fragment thereofcomprises a light chain with the amino acid sequence set forth in SEQ IDNO:
 11. 9. The antibody and/or binding fragment thereof according to anyone of claims 1 to 8, wherein the antibody and/or binding fragmentthereof comprises a heavy chain variable region with the amino acidsequence set forth in SEQ ID NO:12.
 10. The antibody and/or bindingfragment thereof according to any one of claims 1 to 9, wherein theantibody and/or binding fragment thereof comprises heavy chain IgG1 orIgG4 isotypes, with the amino acid sequence of said isotypes set forthin SEQ ID NO: 13 or SEQ ID NO:14.
 11. The antibody and/or bindingfragment thereof of claim 1, wherein the light chain complementaritydetermining region CDR-L3 and heavy chain complementarity determiningregions CDR-H1, CDR-H2 and CDR-H3 have at least 70%, at least 80% or atleast 90% sequence identity to SEQ ID NOS: 1 to 4, respectively.
 12. Theantibody and/or binding fragment thereof of any of claims 1 to 11,wherein the antibody and/or binding fragment thereof is selected fromthe group consisting of a monoclonal antibody, an immunoglobulinmolecule, a Fab, a Fab′, a F(ab)2, a F(ab′)2, a Fv, a disulfide linkedFv, a scFv, a disulfide linked scFv, a single chain domain antibody, adiabody, a dimer, a minibody, a bispecific antibody fragment, a chimericantibody, a humanized antibody and a polyclonal antibody.
 13. Theantibody and/or binding fragment thereof of any one of claims 1 to 12,wherein the αKlotho polypeptide is mammalian αKlotho polypeptide. 14.The antibody and/or binding fragment thereof of claim 13, wherein themammalian αKlotho polypeptide is human αKlotho polypeptide.
 15. Theantibody and/or binding fragment thereof of claim 13, wherein themammalian αKlotho is rodent αKlotho polypeptide.
 16. The antibody and/orbinding fragment thereof of claim 15, wherein the rodent αKlothopolypeptide is mouse αKlotho polypeptide or rat αKlotho polypeptide. 17.The antibody and/or binding fragment thereof of any one of claims 1 to16, wherein the folded αKlotho polypeptide is soluble folded αKlothopolypeptide.
 18. The antibody and/or binding fragment thereof of claim17, wherein the antibody and/or binding fragment thereof binds solublefolded αKlotho polypeptide found in urine, plasma, and/or serum.
 19. Theantibody and/or binding fragment thereof of any one of claims 1 to 18,wherein the antibody and/or binding fragment thereof binds a complexcomprising folded αKlotho polypeptide.
 20. The antibody and/or bindingfragment thereof according to claim 19, wherein the folded αKlothopolypeptide forms a complex with a fibroblast growth factor (FGF)receptor, optionally FGFR1c.
 21. The antibody and/or binding fragment ofany one of claims 1 to 20, wherein the antibody and/or binding fragmentis labeled with a detectable tag.
 22. The antibody and/or bindingfragment of claim 21, wherein the detectable tag is His-tag, a HA-tag, aGST-tag, or a FLAG-tag.
 23. An antibody complex comprising the antibodyand/or binding fragment thereof of any one of claims 1 to 22 andαKlotho, optionally further comprising FGFR1c.
 24. The antibody complexof claim 23 comprising FGFR1c and optionally further comprising FGF23.25. A nucleic acid encoding an antibody and/or binding fragment thereofcomprising a light chain variable region and a heavy chain variableregion, the light chain variable region comprising complementaritydetermining region CDR-L3 and the heavy chain variable region comprisingcomplementarity determining regions CDR-H1, CDR-H2 and CDR-H3, with theamino acid sequences of said CDRs comprising one or more of thesequences set forth below: a. CDR-L3; (SEQ ID NO: 1) X₁X₂X₃X₄PX₅,

wherein X₁ is A or S, X₂ is G or A, X₃ is Y or F, X₄ is S or A, X₅ is Ior V; b. CDR-H1: (SEQ ID NO: 2) X₆X₇X₈X₉X₁₀X₁₁,

wherein X₆ is I or V, X₇ is S or A, X₈ is Y, F or S, X₉ is Y, F or S,X₁₀ is S or A and is I or V; c. CDR-H2; (SEQ ID NO: 3)X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁,

wherein and X₁₂ is Y, F or S, X2₁₃ is I or V, X₁₄ is S or A, X₁₅ is P orS, X₁₆ is S or A, X₁₇ is Y or F, X₁₈ is G or A, X₁₉ is Y or F, X₂₀ is Tor S and X₂₁ is S, A or Y; d. CDR-H3: (SEQ ID NO: 4)X₂₂X₂₃VYX₂₄X₂₅X₂₆X₂₇WX₂₈GX₂₉GM,

wherein X₂₂ is Y or F, X₂₃ is Y or F, X₂₄ is A or S, X₂₅ is S or A, X₂₆is H or N, X₂₇ is G or A, X₂₈ is A or S and X₂₉ is Y or F.
 26. A vectorcomprising the nucleic acid of claim
 25. 27. A recombinant cellproducing the antibody and/or binding fragment thereof of any one ofclaims 1 to 22, comprising the nucleic acid of claim 25 or the vector ofclaim
 26. 28. A composition comprising the antibody and/or bindingfragment thereof of any one of claims 1 to 22, the nucleic acid of claim25, the vector of claim 26 or the cell of claim
 27. 29. An immunoassaycomprising the antibody and/or binding fragment thereof of any one ofclaims 1 to
 22. 30. The immunoassay of claim 29 wherein the immunoassayis an enzyme linked immunosorbent assay (ELISA).
 31. The immunoassay ofclaim 30, wherein the ELISA is a sandwich ELISA.
 32. A method forproducing an antibody and/or binding fragment thereof with specificbinding affinity to an epitope of an αKlotho polypeptide according toany one of claims 1 to 22, the steps comprising: a. expressing in a hostcell a nucleic acid encoding amino acid sequences of the antibody and/orbinding fragment thereof according to any one of claims 1 to 11; b.culturing the host cell to produce the antibody and/or binding fragmentthereof; and c. isolating and purifying the antibody and/or bindingfragment thereof from the host cell.
 33. An assay for measuring level ofαKlotho polypeptide in a sample, the assay comprising: a. contacting asample with the antibody of any one of claims 1 to 22 under conditionsto form an antibody: αKlotho complex; and b. detecting the antibody:αKlotho complex.
 34. An assay for detecting and/or measuring solubleαKlotho the method comprising: a. contacting a sample, the sample beinga biological fluid, with the antibody or binding fragment describedherein under conditions to form an antibody: soluble αKlotho complex;and b. detecting the antibody: soluble αKlotho complex.
 35. The assay ofclaim 33 or 34, wherein the antibody:αKlotho complex is detected byimmunoprecipitation, immunoblot, immunohistochemistry,immunocytochemistry and fluorescence-activated cell sorting (FACS). 36.A method for screening, for diagnosing or for detecting kidney conditionselected from chronic kidney disease (CKD) and acute kidney injury (AKI)in a subject, the method comprising: a. measuring the level of αKlothoin a sample from a subject optionally using an antibody or fragmentthereof of any one of claims 1-22 or using the assay of claim 33 or 34;and b. comparing the level of αKlotho in the sample with a control,wherein a decreased level of αKlotho in the sample compared to thecontrol is indicative that the subject has a kidney condition selectedfrom CKD or AKI.
 37. The method of claim 36, wherein the CKD is earlyCKD, optionally stage 1, stage 2, stage 3, stage 4, stage 5 or stage 6.38. The method of claim 36 or 37 wherein the sample is selected from afresh tissue sample, a frozen sample and a fixed sample such as a mildlyfixed sample.
 39. The method of any one of claims 36 to 38, wherein thelevel of αKlotho is determined by immunoprecipitating.
 40. A method forprognosticating a recovery after AKI, the method comprising: a.determining the level of αKlotho in a sample from a subject; and b.comparing the level of αKlotho in the sample with a control, wherein anincreased level of αKlotho in the sample compared to the control isindicative that the subject has a higher likelihood of recovery afterAKI.
 41. A method for prognosticating long term complications after AKI,the method comprising: a. determining the level of αKlotho in a samplefrom a subject; and b. comparing the level of αKlotho in the sample witha control, wherein an increased level of αKlotho in the sample comparedto the control is indicative that the subject has a higher likelihood offewer long term complications after AKI.
 42. A method forprognosticating the rate of progression of CKD, the method comprising:a. determining the level of αKlotho in a sample from a subject; and b.comparing the level of αKlotho in the sample with a control, wherein anincreased level of αKlotho in the sample compared to the control isindicative that the subject has a higher likelihood of
 43. A method forprognosticating extra-renal complications in CKD, the method comprising:a. determining the level of αKlotho in a sample from a subject; and b.comparing the level of αKlotho in the sample with a control, wherein anincreased level of αKlotho in the sample compared to the control isindicative that the subject has a higher likelihood of having fewerextra-renal complications related to CKD.
 44. A kit comprising anantibody and/or binding fragment thereof according to any one of claims1 to 22, a reference agent and optionally instructions for use thereof.